Evaluation of Antifungal Activity by Mixed Oxide Metallic Nanocomposite against Candida spp.

Round 1

Reviewer 1 Report

The article "Evaluation of Antifungal Activity by Mixed Oxide Metallic 2
Nanocomposite Against Candida spp." is very well presented. The research is interesting and helpful for scientific area. 

Author Response

No coments by the reviewers

Reviewer 2 Report

This is a very nice work from Reyes-Lopez and collaborators which make a comprehensive study of action against Candida SPP by using Zirconia and Silver oxides nanoparticles. I consider that this is a well-done work and could be accepted for publication in this journal after minor revisions according to described below;

• The ideia of studying and applying nanoparticles is interesting but it presents also some limitations in living organisms, in some cases de difficulty of solubilization of such nanoparticles in water for example. Some nanoparticles seems to be very toxic to the live organisms as well as some traditional molecular compounds. I think the authors must compare the results presented in this work with other already published works that use molecular compounds see the recent published works; Journal of Inorganic Biochem. 213 (2020) 111277 and Molecules 2018, 23(8), 1856; I think that these works also presents biological results against Candida SPP. As an innovative work, presenting biological activities of nanoparticles it is fundamental in the discussion part, that the authors make a comparison of biological activities between nanoparticles results and molecular metalorganic compounds.
• The DLS results show that the authors synthesized nanoparticles presenting different sizes from 76 to 14 nm. Is there some experimental evidence of biological behavior by analyzing the size effect of nanoparticles?
• How do the authors make the dispersion or solubilization of nanoparticles before the biological experiments (make it clear in the experimental part). Was used ultrasonic bath in this process?
• Is there in the material supplementary some photo or image of the experiment such as biological for example? Which color the nanoparticles solution present after dissolution and incubation? I could see in the UV-Vis experiment that the prepared nanoparticles are colored. Were the Uv-Vis experiments make on the suspension of nanoparticles?  Which solvent was used? Did the authors make UV-Vis experiments in solid-state like diffuse-reflectance?
• Were the biological experiments made in presence of light or in the darkroom?

Thank you for the contribution to the bioactive anti-Candida compounds.

Author Response

The idea of studying and applying nanoparticles is interesting but it presents also some limitations in living organisms, in some cases the difficulty of solubilization of such nanoparticles in water for example. Some nanoparticles seem to be very toxic to the live organisms as well as some traditional molecular compounds. I think the authors must compare the results presented in this work with other already published works that use molecular compounds see the recent published works;

1. a) Journal of Inorganic Biochem. 213 (2020) 111277 and Molecules 2018, 23(8), 1856; I think that these works also presents biological results against Candida SPP. As an innovative work, presenting biological activities of nanoparticles it is fundamental in the discussion part, that the authors make a comparison of biological activities between nanoparticles results and molecular metalorganic compounds.

- Response: Thank you for your kind suggestions and they are well appreciated. Considering the objective of the research, what we did was to compare our results with previous works published on pure metal or metal oxide and other synthesized mixed metal oxide nanoparticles.

We need more information of suggested article

1. b) The DLS results show that the authors synthesized nanoparticles presenting different sizes from 76 to 14 nm. Is there some experimental evidence of biological behavior by analyzing the size effect of nanoparticles?

- Response: The evidence was presented in the growth kinetics graph in Figure 4 which shows that as the nanoparticles size decreases, the antifungal activity of the samples increases which can be confirmed by the decrease in the growth kinetics and the percentage inhibition.

1. c) How do the authors make the dispersion or solubilization of nanoparticles before the biological experiments (make it clear in the experimental part). Was used ultrasonic bath in this process?

- Response: The nanoparticles were measured and weighed into the microdilution plate and the aliquots containing the fungi was added. Ultrasonic bath was not used.

1. d) Is there in the material supplementary some photo or image of the experiment such as biological for example? Which color the nanoparticles solution present after dissolution and incubation? I could see in the UV-Vis experiment that the prepared nanoparticles are colored.

- There was no supplementary photo or image submitted along with this manuscript. There was no color change on adding the nanoparticles to the aliquot during the biological experiment. The only observable change was the increased turbidity in the solution containing zirconia nanoparticles while the turbidity decreases as the concentration of silver oxide against the nanoparticles increases

1. e) Were the Uv-Vis experiments make on the suspension of nanoparticles? Which solvent was used?

- Response: The UV-Vis experiment was done by the suspension of the nanoparticles in distilled water

1. F) Did the authors make UV-Vis experiments in solid-state like diffuse-reflectance?

Response: The nanoparticles were suspended in distilled water to make the Uv-Vis measurement.

1. g) Were the biological experiments made in presence of light or in the darkroom?

- Response: The aliquots were prepared in the presence of light before incubating for 24 hours and the use of the spectrophotometric machine for the 24 hours growth measurement.

Reviewer 3 Report

The manuscript entitled "Evaluation of Antifungal Activity by Mixed Oxide Metallic Nanocomposite Against CANDIDA SPP." is dealing with antifungal mixed nanoparticles ZrO₂-Ag₂O. The authors have mainly studied the effect of NPs against Candida spp. with interesting results.

However, authors should pay attention to revisions needed to meet the journal's scope and requirements.

The whole manuscript should be checked for typographical mistakes and sent for language editing. Also, the introduction should be rewritten. The length of the introduction shows an enormous disproportion to the small number of used citations.

The authors present their material as an antifungal. However, the experiments were performed only against Candida spp. If we talk about antifungal properties in general, other types of fungi should be tested, such as Aspergillus. Confirmation of these properties is necessary to accept this manuscript.

In experimental section – materials preparation: Insufficiently described purification of nanoparticles. How was the complete removal of the starting precursors ensured?

In experimental section – 2.4.1: What does it mean standardized suspension? What was the exact concentration of fungi in the starter suspension?

In the experimental section – 2.4.2: It is unclear in what form (powder, gel, etc.) the NPs were used in the microdilution plates and why yeasts were added.

Could the authors explain why only one voltage was used in the STEM experiment? Also, there is missing the sample preparation and type of used grid. It would be advisable to make micrographs of all concentrations. They could be added to the supplement. How do the authors explain irregularly shaped nanoparticles with some agglomeration (Fig. 3c)?

The section about Raman spectroscopy is missing few necessary experimental conditions. The power of the excitation laser is missing. If the used power was too high, it could cause substantial heating of the sample. The possible heating of the sample during measurements could influence the uptake of CO₂ on Ag₂O.

Second, what is the strong peak in Fig. 2b),c) emerging on the left-hand side of the graph (possible peak at approx 530 cm^-1)? This peak looks much stronger than those from nanoparticles and is not commented.

XRD and IF are missing in the experimental part.

In section 3.4 (DLS results), it is useless to describe what a method is and for what it is used.

Was the antifungal effect (zone of inhibition) tested only 24h? After how long have been all NPs reduced and their antifungal effect disappeared?

The mechanism of action of the NPs complex is insufficiently explained. It is only a summary of potential mechanisms from several sources. If the effect were to be reflected in ROS production, I suggest that the authors measure this production by a simple EPR method. However, due to ROS's short lifetime and the extreme ability of nutrient media to capture ROS, this possibility is less likely.  It is also known that both types of NPs have an antimicrobial effect. Why do the authors claim that Zr-NPs improved the effect only by their stabilizing function? What synergistic effect do authors expect from this complex? Can it behave, e.g., as an intercalator?

The results lack a more extensive discussion with the scientific literature.

Research on these NPs (as antibacterial material) has already been published in the Dose-Response (https://doi.org/10.1177%2F1559325820941374). The same characterization techniques were used. This manuscript is only an extension of the original article with many errors and inaccuracies and does not bring novelty and originality. On top of that, there is no novel processing technique presented nor improved. Therefore, I do not find this manuscript in the scope of this journal. For this reason, I recommend rejecting the manuscript.

Author Response

1) However, authors should pay attention to revisions needed to meet the journal's scope and requirements.

- Response: The revisions have been done to meet the journal’s scope and requirements.

2) The whole manuscript should be checked for typographical mistakes and sent for language editing. Also, the introduction should be rewritten. The length of the introduction shows an enormous disproportion to the small number of used citations.

- Response: The typographical mistakes have been corrected and edited and the language have been improved. The introduction has been improved and more citations have been included.

3) The authors present their material as an antifungal. However, the experiments were performed only against Candida spp. If we talk about antifungal properties in general, other types of fungi should be tested, such as Aspergillus. Confirmation of these properties is necessary to accept this manuscript.

- Response: The title of the article makes an emphasis on the testing of the nanoparticles on Candida spp. The fact that there are other types of fungi which were not tested in this work, I believe does not make it inappropriate to present the material as an antifungal agent. There are numerous fungi, and all cannot be tested. We only decided to work with fungi of interest

4) In experimental section – materials preparation: Insufficiently described purification of nanoparticles. How was the complete removal of the starting precursors ensured?

- Response: The starting precursors were removed during calcination.

5) In experimental section – 2.4.1: What does it mean standardized suspension? What was the exact concentration of fungi in the starter suspension?

- Response: Standardized suspension is the dilution that contains the needed working concentration. We prepared the solution by putting a colony of the fungus in water. It is from this solution that we calculated the optical density of the solution to match the working concentration of 1.3 X10⁶ CFU/mL.

6) In the experimental section – 2.4.2: It is unclear in what form (powder, gel, etc.) the NPs were used in the microdilution plates and why yeasts were added.

- Response: It was stated that 0.02 mg (powder) of each of the nanoparticles was measured and were plated in the microdilution plate. The yeast was added to analyze the interaction between each of the nanoparticles and the different tested yeast.

7) Could the authors explain why only one voltage was used in the STEM experiment? Also, there is missing the sample preparation and type of used grid. It would be advisable to make micrographs of all concentrations. They could be added to the supplement. How do the authors explain irregularly shaped nanoparticles with some agglomeration (Fig. 3c)?

- Response: It is because it is a comparative study of the different samples as such, it is expected to use the same voltage and scale for all the samples to be able to make a good comparison.

- To carry out the STEM analysis, the samples are placed on the STEM disc and placed in the STEM machine for measurement.

- Irregularly shaped means that there is no dominant shape. It has part of different shapes.

8) The section about Raman spectroscopy is missing few necessary experimental conditions. The power of the excitation laser is missing. If the used power was too high, it could cause substantial heating of the sample. The possible heating of the sample during measurements could influence the uptake of CO₂ on Ag₂O.

- Response: Full experimental information has been added.

1. b) Second, what is the strong peak in Fig. 2b)

- Response: 565 Raman shift

1. c) emerging on the left-hand side of the graph (possible peak at approx. 530 cm^-1)? This peak looks much stronger than those from nanoparticles and is not commented.

- Response: It is not a peak, but a descending line that can also be seen in Figure 2c.

9) XRD and IF are missing in the experimental part.

- Response: The information has been added

10) In section 3.4 (DLS results), it is useless to describe what a method is and for what it is used.

- Response: It has been corrected and edited.

11) Was the antifungal effect (zone of inhibition) tested only 24h? After how long have been all NPs reduced and their antifungal effect disappeared?

- Response: The antifungal activity was determined after 24 hours according to the protocol of the Clinical Laboratory Standard Institute (CLSI) and the European Committee on Antibiotic Susceptibility Study (EUCAST)

12) The mechanism of action of the NPs complex is insufficiently explained. It is only a summary of potential mechanisms from several sources. If the effect were to be reflected in ROS production, I suggest that the authors measure this production by a simple EPR method. However, due to ROS's short lifetime and the extreme ability of nutrient media to capture ROS, this possibility is less likely.  It is also known that both types of NPs have an antimicrobial effect. Why do the authors claim that Zr-NPs improved the effect only by their stabilizing function? What synergistic effect do authors expect from this complex? Can it behave, e.g., as an intercalator?

- Response: This research work was not to investigate the mechanism of action of nanoparticles, that was why we cited articles that have reported on the mechanisms of actions of nanoparticles to buttress our discussion.

At the concentration at which we synthesized Zirconia nanoparticles; they did not record any inhibition.

The results lack a more extensive discussion with the scientific literature.

13) Research on these NPs (as antibacterial material) has already been published in the Dose-Response (https://doi.org/10.1177%2F1559325820941374). The same characterization techniques were used. This manuscript is only an extension of the original article with many errors and inaccuracies and does not bring novelty and originality. On top of that, there is no novel processing technique presented nor improved. Therefore, I do not find this manuscript in the scope of this journal. For this reason, I recommend rejecting the manuscript.

- Response: The previously published article was about the antibacterial property of the synthesized nanoparticles while this manuscript is reporting on the antifungal property of the synthesized nanoparticles.

The raman and UV-visible analyses have not been reported nor published earlier.

The errors and inaccuracies have been corrected and edited.

The objective of this research work was not to report a novel synthetic method or improved processing technique but to investigate the antimycotic effect of ZrO₂ and Ag₂O nanoparticles against selected pathogenic Candida species causing life threatening fungal infections in humans and to evaluate the changes in the antimycotic activity of Ag₂O on stabilizing with ZrO₂ nanoparticles. Not all research work is done to report novel technique or method.  

Reviewer 4 Report

This manuscript reported the synthesis, characterization and antifungal action of mixed oxide metallic nanocomposite. Article is well organized, while suffers from many necessary things at few instances. This reviewer rather likes to go for a minor revision before accepting this for publication in Processes.

Following minor points should be corrected:

1. Keywords, Line 25, ZrO₂-Ag₂O, Candida
2. Line 347, fig. 5 should be corrected. Line 344 and so on.
3. Candida is right. Please note “Italic” for test strains. For example, Line 338, 346, 350, and so on.
4. Spaces, Line 384, In vitro
5. Line 123, Subscript for ZrO2-Ag2O.

Author Response

1) Keywords, Line 25, ZrO₂-Ag₂O, Candida

- Response: Candida has been corrected and placed in italics and ZrO₂-Ag₂O has been corrected.

2) Line 347, fig. 5 should be corrected. Line 344 and so on.                                                      

- Response: All have been edited and corrected

3) Candida is right. Please note “Italic” for test strains. For example, Line 338, 346, 350, and so on.

- Response: All have been edited and corrected

4) Spaces, Line 384, In vitro

- Response: It has been edited and corrected

5) Line 123, Subscript for ZrO2-Ag2O.

- Response: It has been edited and corrected

Reviewer 5 Report

This work evaluated the antifungal activity of ZrO2-Ag2O nanoparticles, and found that these nanoparticls had a excellent antibacterial activity and non-toxic effect. Two main issues need be improved as follows:

1. What is so-called enhanced stability? It is not clear in the manuscript.
2. In general, oxide of Ag has some degree of toxicity, but in this work, it is non-toxic. please give more explanation in the discussion part.

Author Response

1) What is so-called enhanced stability? It is not clear in the manuscript.

- Response: Previous literatures have reported that the lack of stability of Ag and Ag₂O is responsible for agglomeration which results in decreased antibacterial activity. Therefore, mixing silver oxide with zirconia helps to prevent agglomeration because the zirconia nanoparticles helps to confer stability on silver oxide by reducing the high surface energy of silver oxide.

2) In general, oxide of Ag has some degree of toxicity, but in this work, it is non-toxic. please give more explanation in the discussion part.

- Response: One of the ideas of the research work was to use zirconia nanoparticles at a concentration that it will neutralize the toxicity of silver oxide nanoparticles to human cells by taking advantage of the non-toxic nature of zirconia nanoparticles