Antifungal Properties of Zinc Oxide Nanoparticles on Candida albicans

Round 1

Reviewer 1 Report

In my opinion, the work was done at a high level, but the conclusions should be improved.

The authors thank the reviewer for the thoughtful comments and suggestions about our work. We have revised the conclusion part and we hope that revision is fine with you. Thank you so much again Professor.   

Reviewer 2 Report

1. The authors reported a study of anti-fungal properties of ZnO nanoparticles. The referee believe that the authors should address following issues before publication:

The authors thank the reviewer for the thoughtful comments and suggestions about our work. We hope that the revised version has addressed all your comments. 

2. Table 1: An EC₅₀ value should be provided.

Authors thank the reviewer to pointing this out. As per the reviewer suggestion Half maximal effective concentration (EC₅₀) value is reported in the revised manuscript (page No.6, line No. 186-188)

Based on the reported percentage of grow inhibition on C. albicans by the treatment of ZnO NPs, EC₅₀ value for ZnO NPs against C. albicans was reported to be 35.59 μg/mL.

and the supporting discussion is added in page No. 12, line No. 300-302,

Further, our results reported a lower EC₅₀ value for ZnO NPs (35.59 μg/mL) on C. albicans as compared with 131 mg/L as the EC₅₀ of ZnO NPs against Saccharomyces cerevisiae at 24 h, indicating a higher antifungal potential of ZnO NPs on C. albicans [34].

3. Figure 7 and Table 2: The authors would like to use the shift in IR spectrum to support the claim that the ZnO nanoparticles binds with the yeast cell walls. However, the shift of peaks may also rise from the presence of ZnO Nanoparticles. Also, the extract from a living yeast cell (negative control) and an apoptosed cell can be different, but this is not necessarily related to binding of ZnO Nanoparticles to cell walls (for example, Plos One 2017; 12(7): e0180680). The referee suggests that the IR Spectrum of ZnO nanoparticles should be plotted together, and above-mentioned factors should be discussed.

The authors thank the reviewer for the insightful comments and suggestions about our work. We have included the FTIR data of ZnO NPs in the revised version (page No. 9, line No. 241-243).

The FTIR spectrum of ZnO nanopowder showed peaks at 3425, 1628 and 524 cm^{- 1}. Earlier studies have reported stretching of O-H vibrations between 3400 and 3600 cm⁻¹[29], Zn-O stretching at 1634.00 cm⁻¹[30], stretching of ZnO NPs at the range of 400–800 cm^−1.

Big thanks for your comment on the peak shift in the FTIR spectrum due to apoptosis. This is a new information we found through your comment. It’s interesting to know that peaks can be shifted due to the damage or destruction of biomolecules in the cell during apoptosis process according to Mihoubi, Wafa, Emna Sahli, Ali Gargouri, and Caroline Amiel. "FTIR spectroscopy of whole cells for the monitoring of yeast apoptosis mediated by p53 over-expression and its suppression by Nigella sativa extracts." PLoS One 12, no. 7 (2017): e0180680.

However, no specific peak shift in the apoptosis is related to our FTIR results. Notably, the peak shift from1048 to 1076 cm^-1 reported in that study can be considered may be due to the oxidative damage of DNA of yeast cells. But in our study the peak shift is reported from 1076 (in control, healthy cells) and 1048 (in treatment).Hence, here we would not say DNA damage happened in the healthy cells before the exposure to ZnO NPs .

4. Line 387~388: The authors claimed that the binding of ZnO nanoparticles was through electrostatic interactions between Zn²⁺ and anion groups on the yeast cell walls. The referee believes that given the scale of the system investigated (a few hundreds of nanometers to a few micrometers), if there are electrostatic interactions between ZnO nanoparticles and yeast cell walls, it is the surface charges of ZnO nanoparticles and yeast cell walls that should be considered, not the charges of individual atoms/functional groups. The referee suggests that the authors should measure the surface charges of particles (e.g., zeta-potential or electrophoresis) to explain the electrostatic interactions between ZnO nanoparticles and yeast cell walls.

The authors thank the reviewer for the thoughtful comments and suggestions about our work and these remarks help us to improve the quality and visibility of the manuscript. In this present study, we did not take zeta-potential analysis of ZnO nanoparticles since we don’t have the facility for zeta potential measurement. However, we will do the zeta potential through outsourcing in our future reports.

We have included the mechanisms of cellular accumulation of nanoparticles  in the revised version based on the earlier study results based on the earlier reports (page no. 12 -13, line No. 334-353) which mainly suggest the involvement of an attractive van der Waals force, kinetic and thermodynamic exchanges and adhesion which exist between the surface of nanoparticles and the surface biomolecules of the microorganisms.

Further, the surface accumulation of ZnO NPs on the yeast cell is evidently demonstrated by the EDX analysis of the present study (Figure 8). To explain the reason behind the accumulation of ZnO NPs on the surface of the yeast cells and the subsequent cell damage or death, it is important to consider the composition of the fungal cell wall which is composed of glycoproteins, polysaccharides (especially glucan and chitin), and the hydrophobins and amphipathic proteins which are the important proteins in fungai that are involved in the interaction with their environments[44, 45]. Up on the exposure of fungal cells to the ZnO NPs,  dynamic physicochemical interactions, kinetic and thermodynamic exchanges, an attractive van der Waals force would act between the NPs surface occur between the surface of the NPs and the surface of the biological component such as membrane and proteins. All these interactions are believed to cause interaction of ZnO NPs on the cell membrane of fungal cells, leading to the acumulation of NPs on the cell membrane and the subsequent membrane disruption [46, 47]. Further, the process of adhesion of NPs on the surface of the microorgamism is also believed to be an important reason for cytotoxicity. The surface biomolecules of microorganiams can dominate in cell adhesion process bacause of their functional groups, highly charged structure, and the bridging effect with the surface of nanoparticles. Important biomolecules such as lipopolysaacharides, and phospholipids from the cell surface were found to adsorb on ZnO NPs surfaces during NP exposure, leading to the structural changes in proteins and phospholipids which are the more likely reason for the cytotoxicity of microorganisms in addition to membrane damage [48-50].

 Many thanks Professor, your constructive comments really  made us to learn many new information.     

Reviewer 3 Report

The authors of the manuscript titled: "Antifungal Properties of Zinc Oxide Nanoparticles on Candida albicans" present a somewhat routine investigation of antifungal properties of ZnO nanoparticles. A quick review shows number of studies on the antifungal properties of ZnO nanoparticles against Candida albicans. Therefore, the novelty and significance of the content of this manuscript is in question. 

1. The introduction section of the manuscript is lengthy, yet a very basic and surface-level summary of the problem. You should rewrite this section to be more concise and, more importantly, you should state the objectives of the work clearly here, including the novelty of the performed experiments. You then need to comment on in the Conclusions part how the obtained results expand on the state of the art described in the introduction section.

The authors thank the reviewer for the thoughtful comments and suggestions about our work. We have reduced the introduction part by removing the non- relevant contents to make it concise. To our knowledge, we believe the following are the novelty of the present study; morphological damage by ZnO NPs through SEM, the cellular accumulation of ZnO NPs and the functional groups involved in the surface interaction of ZnO NPs through FTIR in of the Candida albicans (ATCC 1023) 

The objectives and the novelty of the study are included in the revised version more concisely as below:

Hence, the present study was aimed to determine the antifungal effect on the dose-dependent mechanism by exposing the C. albicans with a wide range of concentrations of ZnO NPs from 5, 10, 20, 40, 80, and 160 µg/mL. The antifungal effects were evaluated using the turbidity method, iodonitrotetrazolium chloride (INT) assay and colony count method. Besides, the study also investigated the involvement of functional groups from the cell wall of C. albicans in the interaction of ZnO NPs on the cell surface as well as the morphological changes caused by ZnO NPs on C. albicans.

2. The discussion section does not need to include the results already stated (the first part of the chapter 4.1), however, you should include comparisons with results obtained from literature. You need to add specific relevant quantitative and qualitative results from literature and compare those to the results achieved in this work. The chapter 4.2 seriously needs to be reworked to be better readable, add paragraphs and structure the text so it is better accessible by the readers. You have to improve the overall the style and language of the text significantly. There are numerous grammatical mistakes and the text is overall difficult to read due to lack of structure. I think the above-mentioned problems with the manuscript lead me to ask the authors to perform major revisions of the manuscript if it is to be published in this journal.

The authors thank the reviewer for the thoughtful comments and suggestions about our work. The discussion part has been revised completely based on your comments by supporting and comparing with the results obtained from the literatures. The whole manuscript has been thoroughly checked for the grammatical and spelling mistakes and corrected them all in the revised version.

We hope that our revision version has addressed all your comments and suggestions to improve the manuscript readability and quality. Thank you so much again Professor.

Round 2

Reviewer 1 Report

Good job

Reviewer 2 Report

The revised manuscript is suitable for publication.

Thank you so much for your review and acceptance of your reply in the revised version earlier and approving our manuscript as suitable for submission. 

The attached is the latest revision on the corrected sentence and grammatical errors pointed out by the other reviewer. 

Thank you again 

Reviewer 3 Report

The authors have improved on the structure of the text significantly. The manuscript is now more readable and the goals and results of the work are stated clearly. I have only several minor further suggestions for improvement:

The sentence on lines 82-85 should be moved to the previous paragraph.

The sentence on lines 56-57 is gramatically incorrect and needs to be rewritten.

Line 330 - the text "of involvement" is redundant and should be erased.

It would be clearer if  the lines in Fig. 6 (FTIR spectrum) were described by an in-picture legend rather than just the caption itself.

We are really grateful to your commitment in thoroughly reviewing our revised manuscript and giving your feedback on the grammatical errors, rearranging the texts and labelling the legends in the figure for better readability of our manuscript.  Thank you so much. 

We have done all the suggested changes as below.

1. The sentence on lines 82-85 should be moved to the previous paragraph.

Moved to line no 78-80. and stimulates the overproduction of reactive oxygen species (ROS), such as hydroxyl group, superoxide anions radicals and hydrogen peroxide, in the cells which can lead to cell death. 

2. The sentence on lines 56-57 is gramatically incorrect and needs to be rewritten.

Rewritten as below

The therapeutic alternatives for treating fungal infections are quite limited comparing with the available antibacterial agents

3. Line 330 - the text "of involvement" is redundant and should be erased.

Erased. 

4. It would be clearer if  the lines in Fig. 6 (FTIR spectrum) were described by an in-picture legend rather than just the caption itself.

The figure 6 is labelled accordingly in the revised version. 

Round 3

Reviewer 3 Report

All the stated issues were corrected, the manuscript is now acceptable for publication.