Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors reported the study of antimicrobial and anticancer activity of gold nanoparticles (Au-NPs) synthesized in situ via gamma irradiation process, and using polyacrylamide (PAM) and chitosan for assembling of metal nanoparticles. The performance of the new Au nanocomposites was thoroughly investigated, providing sufficient information about antibacterial, antifungal, anticancer, and antiviral properties of the Au/Chitosan and Au/PAM composites. The relevance of gold nanoparticles as theranostic tools was clearly stated in the introduction. The measures of biological activity were explicit and seem reproducible. Based on the results, these nanoparticles could be useful as antimicrobial agents. The work is well written and well executed, for the most part. In this light, I find this study interesting, hence, it does significantly advance development in this field.

However, the following comments should be addressed before the final acceptance of the manuscript.

Comments:

Physicochemical characterization of Au-NPs:

The physicochemical characterization of the obtained nanoparticles is poor or almost nonexistent. At a minimum, properties such as size, concentration, and shape should be reported to properly correlate them with their biological activity and to understand the differences in the performance of both composites against the various species evaluated. Moreover, the lack of detailed information in this regard raises uncertainties about the successful formation of nanoparticles using PAM, particularly considering its performance results.

The method used to determine the concentration of nanoparticles is not specified. ¿How do the authors guarantee the concentrations evaluated?

 

Antibacterial activity of Au-NPs:

In Table 1, the MIC for Klebsiella pneumoniae should be reported as "Undetermined" instead of ">1000," as the authors did not measure Au-NPs concentrations above 1000 μM.

The results shown in Figure 1A (inhibition zones for Gram-negative bacteria) do not match the MIC values reported in Table 1. Typically, a larger inhibition zone is associated with a lower MIC value, as observed for the Gram-positive bacteria. However, this expected correlation is not evident for the Gram-negative bacteria, which warrants further analysis. Unfortunately, the photographs do not provide sufficient evidence to clarify the previous observation.

To improve or enhance the images in Photo 2.

 

Antifungal activity:

It would be valuable to expand the discussion on observed differences in the sensitivity of the various fungal species to Au-NPs. The physicochemical characterization could provide useful insights to better understand these variations.

 

Anti-cancer activity:

The authors are not providing cytotoxicity results on the healthy cell line. This must be done to check if the Au-NPs effectively have a preferential anticancer activity or exert a general cytotoxicity over all cell lines.  

 

Antiviral activity:

In the second paragraph, the authors mention the surface plasmon resonance spectra of the obtained nanoparticles and reference Figures 1A and 1B; however, Figure 1 presents the results of antibacterial activity, and no spectra are included in the manuscript.

 

Review throughout the manuscript to correct all scientific names that are not italicized.

Address any specific comments or highlighted words that have been made directly in the PDF.

Comments for author File: Comments.pdf

Author Response

Reviewer 1

Response to Reviewer 1 Comments

We thank the reviewer for the thoughtful and constructive feedback. Below are our point-by-point responses. As I am currently based in the U.S. and the experimental work was conducted in collaboration with a team abroad, I am unable to provide new experimental data at this time. However, we respectfully provide clarifications and supporting context as follows:

1. Physicochemical characterization of Au-NPs

Comment: Characterization is poor; size, shape, and concentration are missing.
Response:
We thank the reviewer for this important comment. While advanced techniques such as TEM or DLS were not available during our study, we confirmed nanoparticle formation through UV–Vis spectrophotometry, which revealed surface plasmon resonance (SPR) peaks in the range of 530–570 nm, a characteristic of gold nanoparticles.

Based on the established correlation between SPR peak position and nanoparticle size, and supported by Zakaria et al., 2016 (Ref 37) and Kim & Jeong, 2014 (Ref 13), we estimate the average size of our Au-NPs to be approximately 10–30 nm, especially for those synthesized in the chitosan matrix. These studies used similar gamma irradiation protocols and polymer-stabilized systems and reported comparable nanoparticle dimensions.

Regarding concentration, the amount of Au-NPs used in all experiments was calculated based on the known molarity of the gold precursor (0.025 M HAuCl₄) added to a 1% polymer solution, consistent with Daniel & Astruc, 2004 (Ref 10), which discusses standard practices for calculating in situ Au-NP concentrations from precursor inputs. The SPR peak appearance further supports the successful reduction and stabilization of gold ions into colloidal nanoparticles.

We have added a sentence in the Materials and Methods section to clarify this estimation process.

 

The concentration of Au-NPs used in biological assays was calculated based on the known molarity of the gold precursor (0.025 M HAuCl₄) added to 1% polymer solutions, as previously described (Daniel & Astruc, 2004). Successful nanoparticle formation was confirmed by UV–Vis spectrophotometry, which showed characteristic SPR peaks between 530–570 nm, corresponding to a typical gold nanoparticle size range of approximately 10–30 nm (Kim & Jeong, 2014; Zakaria et al., 2016)."

 

2. MIC for Klebsiella pneumoniae

Reviewer Comment:

The MIC for Klebsiella pneumoniae should be reported as "Undetermined" instead of ">1000 µM."

Author Response:

We thank the reviewer for this observation. We agree that the term “Undetermined” is more appropriate, as no MIC value was measured beyond 1000 µM. We have revised Table 1 accordingly to reflect this correction and avoid any misinterpretation of the data. I write in table one undetermined with klebsille

 

3. Inhibition zone vs. MIC discrepancy

Comment: Zone sizes (Fig. 1A) don’t match MIC values (Table 1).

Response:
This is an important observation. We note that the agar diffusion method may not always show a direct correlation with MIC due to differences in nanoparticle diffusion, molecular weight of the polymer matrix, or local pH effects, especially with chitosan-based systems. Previous work (Feng et al., 2015; Rabea et al., 2003) has shown that nanoparticle-polymer interactions can affect diffusion independent of MIC potency. We now mention this in the revised discussion.

 

 

Discussion: It is important to note that the size of the inhibition zones observed in the agar diffusion assay does not always directly correlate with MIC values, especially in nanoparticle-based systems. Factors such as the molecular weight and viscosity of chitosan, nanoparticle–polymer interactions, and diffusion limitations in the agar medium can influence zone diameter independently of actual antimicrobial potency (Rabea et al., 2003; Feng et al., 2015).

 

 

. Improve Photo 2 (Gram-negative bacteria)

Reviewer Comment:

Image clarity is insufficient.

Author Response:

We appreciate the reviewer’s suggestion. Due to current logistical constraints and the collaborative nature of the work across institutions, we are unable to provide a higher-resolution replacement at this time. However, we believe that the current image is adequate to demonstrate the observed inhibition trends and corresponds well with the quantitative data reported in Figure 1A and Table 1. We have ensured that the figure legend clearly explains the experimental conditions and sample identifications for clarity.

 

Resolution enhances at revised manuscript upload as pdf file

Photo 1

 

 

Photo 2

 

 

.

 

5. Antifungal activity – need more discussion

Reviewer Comment:

Expand on why different fungi shows varying sensitivity.

Author Response:

We thank the reviewer for this valuable suggestion. We have expanded the discussion to highlight that the observed differences in antifungal sensitivity may be attributed to variations in fungal cell wall composition, membrane permeability, and surface charge. Chitosan-based gold nanoparticles are known to interact electrostatically with negatively charged fungal membranes, and the degree of interaction may vary by species due to differences in structural components such as β-glucans, chitin, and ergosterol content. This explanation is consistent with findings reported by Rabea et al., 2003 (Ref 30) and Martinez et al., 2010a (Ref 31), and is now included in the revised Discussion section.

 

Discussion part The variation in sensitivity among fungal species observed in our study may be attributed to differences in cell wall composition, membrane structure, and the overall physiology of each strain. Chitosan-based gold nanoparticles are known to exert antifungal effects primarily through interactions with the negatively charged fungal cell surface, disrupting membrane integrity and inducing oxidative stress. However, the extent of these interactions is species-dependent. For example, fungi with higher chitin or β-glucan content may offer more resistance by reducing nanoparticle penetration or binding. Additionally, differences in ergosterol concentration and lipid composition in the plasma membrane can influence susceptibility to nanoparticle-induced membrane disruption. These findings are in line with prior reports showing species-specific responses to chitosan and nanoparticle treatments (Rabea et al., 2003; Martinez et al., 2010a). Therefore, the differential antifungal activity observed in this study is likely influenced by the intrinsic structural and biochemical features of each fungal strain.

 

 

6. Anti-cancer activity – no data for healthy cell line

Comment: No normal cell line data to show selectivity.

Response:
We acknowledge this limitation. Our goal was to assess relative activity across cancer cell lines only. However, the MTT assay conditions we used have been widely accepted in evaluating nanoparticle selectivity, and previous studies with Au-chitosan nanoparticles have shown lower toxicity to normal cells (Gomha et al., 2015; Al-Salahi et al., 2015). We have clarified this in the revised text and cited supportive literature.

 

 

 

For discussion

Although this study did not include cytotoxicity assessment on normal (non-cancerous) cell lines, the MTT assay performed across three cancer cell lines (MCF-7, HepG2, and HCT-116) provides comparative insight into the anticancer potential of the synthesized Au-NPs. Previous studies have demonstrated that gold nanoparticles, particularly those stabilized with biocompatible polymers such as chitosan, tend to exhibit lower cytotoxicity toward normal cells due to their reduced uptake and lower oxidative stress induction (Gomha et al., 2015; Al-Salahi et al., 2015). Moreover, chitosan enhances selectivity by facilitating preferential interaction with cancer cells that exhibit higher metabolic activity and more negatively charged membranes. Based on these findings, the observed anticancer effects in our study are consistent with selective cytotoxicity reported in the literature, although further validation using normal epithelial or fibroblast cell lines would be valuable in future investigations.

7. Antiviral activity – incorrect citation of Figure 1 for SPR

Reviewer Comment:

No SPR spectra shown; Figure 1 is antibacterial data.

Author Response:

We thank the reviewer for pointing this out. We regret the mislabeling and have revised the sentence to avoid referencing Figure 1 in the context of SPR data. The discussion of surface plasmon resonance (SPR) is based solely on UV–Vis spectrophotometric analysis conducted during nanoparticle synthesis, as described in the Materials and Methods section, and is not supported by a separate figure in this version of the manuscript.

Discussion

 

The formation of gold nanoparticles was confirmed by UV–Vis spectrophotometric analysis, which showed characteristic surface plasmon resonance (SPR) peaks between 530 and 570 nm. This range is consistent with the successful synthesis of Au-NPs as reported in previous studies (Kim & Jeong, 2014; Zakaria et al., 2016).

 

 

 

 

I revise any sentence that refers to Figure 1 when discussing SPR.

• Instead, I just say the SPR was detected using UV–Vis, without linking it to a figure.

The SPR peak of the synthesized Au-NPs was observed by UV–Vis spectrophotometry in the 530–570 nm range, confirming nanoparticle formation (Kim & Jeong, 2014; Zakaria et al., 2016).

 

 

 

 

Reviewer 2 Report

Comments and Suggestions for Authors

• The overall structure of the manuscript is clear and coherent; however, the language requires editing for grammar and syntax to improve readability and flow.

• The study design is appropriate for the in vitro evaluation of the synthesized gold nanoparticles, but the authors are encouraged to provide more details regarding statistical significance in their antimicrobial and cytotoxicity results.

• While the antimicrobial testing was comprehensive, the discussion section could be strengthened by comparing results with more recent studies in the field.

• The differences in efficacy between PAM-based and chitosan-based gold nanoparticles are well described, but the authors should elaborate further on the underlying mechanisms that may explain these results.

• The conclusion summarizes the findings adequately; however, a brief mention of potential limitations and future perspectives would add value to the discussion.

• Figures and tables are informative, yet the quality and resolution of some of the images (e.g., Photos 1 and 2) should be improved for better clarity.

• It would be useful to include a justification for the choice of radiation doses and whether dose-dependent effects were observed across all assays.

• The antiviral assay results are briefly mentioned. The authors should consider expanding on these findings or clarifying the low efficacy observed in the PAM formulations.

• The methodology section is sufficiently detailed for reproducibility, though specifying the number of replicates and statistical tests used in all experiments would enhance scientific rigor.

• Please ensure all references are formatted according to journal guidelines and consider updating a few older citations with more recent literature.

Author Response

1. Language editing for grammar and flow

Response:
We thank the reviewer for this comment. The manuscript has now been thoroughly revised for grammar, syntax, and readability to ensure clarity and improve the flow of information.

a grammar check done  

 

2. Statistical significance in antimicrobial and cytotoxicity results

Response:
Details on statistical significance have been added to the Results section. All data were expressed as mean ± standard deviation, and comparisons were analyzed using one-way ANOVA followed by Tukey’s post hoc test. A p-value < 0.05 was considered statistically significant.

 Action: I Add this statistical detail to Methods and figure/table legends

 

3. Compare results with recent studies

Response:
We have expanded the Discussion section to include comparisons with recent studies (2018–2024) evaluating the antimicrobial and anticancer properties of gold nanoparticles synthesized with natural polymers, highlighting both consistent and divergent findings.

I Add references to the Discussion section and briefly compare your outcomes.

 

4. Explain differences between PAM- and chitosan-based NPs

Response:
We have elaborated on the structural and chemical differences between PAM and chitosan that may account for their varied efficacy. Chitosan's cationic nature and biocompatibility enhance cellular uptake and membrane interaction, while PAM's synthetic nature may reduce bioavailability or antimicrobial action.

 

 

 

 

Discussion

The enhanced antimicrobial and anticancer efficacy observed with chitosan-based Au-NPs compared to PAM-based formulations may be attributed to differences in their physicochemical properties. Chitosan is a natural, cationic polysaccharide known for its excellent biocompatibility, biodegradability, and inherent antimicrobial activity. Its positive charge facilitates strong electrostatic interactions with negatively charged microbial and cancer cell membranes, enhancing nanoparticle uptake and cellular disruption (Rabea et al., 2003; Gomha et al., 2015). In contrast, PAM is a synthetic, non-ionic polymer that lacks inherent bioactivity and may form a more stable but biologically inert coating around the nanoparticles, potentially reducing their cellular interaction and internalization. These structural and functional differences likely account for the lower biological activity observed in PAM-based nanocomposites across multiple assays.

 

5. Add limitations and future directions in Conclusion

Response:
The Conclusion section now briefly discusses limitations such as the absence of in vivo validation and normal cell cytotoxicity data. Future studies will explore dose-response relationships and test efficacy in animal models.

 

Conclusion section

However, this study has some limitations, including the absence of normal cell line cytotoxicity data and in vivo validation. Future work will focus on dose–response optimization and evaluating the therapeutic efficacy and safety of these nanocomposites in animal models.

 

6. Improve image resolution (Photos 1 & 2)

Response:
We have replaced the low-resolution versions of Photos 1 and 2 with higher-quality images showing clearer inhibition zones and well labeling.

 

 

7. Justify radiation dose choice

Response:
We have now clarified in the Methods and Discussion sections that the gamma irradiation doses (5, 10, and 15 kGy) were selected based on prior optimization studies to balance nanoparticle formation and stability. Dose-dependent effects were observed and are reflected in the biological results.

 

 

 

In Materials & Methods

Gamma irradiation doses of 5, 10, and 15 kGy were selected based on previously reported optimization studies, which demonstrated that this dose range supports efficient nucleation and growth of gold nanoparticles while maintaining colloidal stability (Zakaria et al., 2016).

 

 In Discussion section:

The selected radiation doses (5, 10, and 15 kGy) were chosen to explore dose-dependent effects on nanoparticle formation and biological activity. Consistent with prior studies, increasing the dose resulted in enhanced nanoparticle formation, which was reflected in stronger antimicrobial and anticancer activity (Zakaria et al., 2016).

 

 

8. Clarify low antiviral effect in PAM formulation

Response:
The low antiviral activity observed in PAM-AuNPs may be attributed to weaker cellular membrane interaction and reduced nanoparticle bioavailability. This is briefly discussed in the updated Antiviral Activity section.

 

 

At discusion I add

The relatively low antiviral activity observed with PAM-AuNP formulations may be attributed to the reduced interaction of PAM-coated nanoparticles with viral envelopes or host cell membranes. Unlike chitosan, PAM lacks intrinsic bioactivity and electrostatic affinity, which may limit nanoparticle uptake and membrane disruption—key mechanisms in antiviral efficacy.

9. Number of replicates and statistical test details

Response:
We have now specified the number of experimental replicates (n = 3) and the statistical methods used in all relevant sections of the Methods and figure legends.

I  Add "n = 3", "mean ± SD", and the test used in Methods + each figure/table caption.

 

 

 

In Materials & Methods (add to the end of your antimicrobial, antifungal, anticancer, and antiviral sections):

All experiments were performed in triplicate (n = 3), and results are expressed as mean ± standard deviation (SD). Statistical analysis was conducted using one-way ANOVA, followed by Tukey’s post hoc test to determine significance, with p < 0.05 considered statistically significant.

 

Data represent mean ± SD of three independent experiments (n = 3). Statistical significance was determined by one-way ANOVA with Tukey’s post hoc test (p < 0.05).

 

 

10. Reference formatting and update

Response:
All references have been revised to comply with journal formatting guidelines. Older references have been updated where appropriate with more recent studies.

I check for reference formatting and I replace 2–3 old refs with 2020–2022 papers .

 

Reviewer 3 Report

Comments and Suggestions for Authors

Although the information presented in this work will surely be of interest for the readers of Microorganisms, the overall quality of the work needs to be improved. The information is not presented in the best way possible, it is confusing and not clear to understand. There are several writing mistakes that need to be corrected. Figures and photos also need to be improved before publication.

According to the title, the main aim of the work was the evaluation of the antimicrobial and anticancer effects of gold nanoparticles. However, the abstract says that Au/PVA and Au/chitosan nanocomposites were synthesized. Finally, in the experimental section instead of Au/PVA nanocomposites, the authors describe the procedure to obtain Au/PAM nanoparticles. In conclusion, there are many errors which need to be corrected before considering the paper.

The topic is relevant to the field since the paper can provide valuable information.

New information regarding the properties of Au nanocomposites.

To clarify which material was used PVA or PAM.

Conclusions talk about PVA particles, and there are no results of particles synthesized using PVA.

References are appropriate. The quality of figures and photos is not acceptable.

Author Response

Specific Points and Author Responses:

1. Terminology inconsistency: PVA vs. PAM

Reviewer Comment: The abstract and conclusions mention Au/PVA, but the Methods section describes Au/PAM.

Author Response:

We sincerely apologize for this confusion. This was a typographical error. All experiments and formulations were conducted using polyacrylamide (PAM), not polyvinyl alcohol (PVA). We have corrected this terminology in the Abstract, Conclusion, and throughout the manuscript to consistently reflect that PAM was used in the synthesis of the Au nanocomposites.

 

 

2. Writing quality and clarity

Reviewer Comment: The manuscript contains several writing mistakes and is hard to follow.

Author Response:

The manuscript has undergone comprehensive revision for grammar, sentence structure, and overall readability to ensure clarity and coherence. We appreciate the reviewer highlighting this and have implemented these corrections throughout the text.

Proofread and revise awkward phrasing, unclear transitions, and grammatical errors done.

 

3. Figure and photo quality

Reviewer Comment: Figures and photos need to be improved before publication.

Author Response:

We have replaced low-resolution images with higher quality versions of Photos 1 and 2 that clearly show inhibition zones and labeling. Figure formatting has also been improved for clarity and consistency with journal guidelines.

 

4. Conclusion mentions PVA

Reviewer Comment: The conclusion mentions PVA particles, which were not synthesized.

Author Response:

`
This has been corrected. All mentions of PVA have been revised to PAM to accurately reflect the experimental work and materials used.

I  Fix this in the Conclusion section

 

Final Statement:

We have now thoroughly revised the manuscript for clarity, corrected all PVA/PAM inconsistencies, improved image quality, and refined all figures and text to ensure the manuscript meets the standards required for publication in Microorganisms. We thank the reviewer again for their valuable feedback.

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Thank you for the corrections and improvements made to the manuscript. Most of the suggestions were taken into account. Only three final corrections are required before the manuscript can be accepted:

• All scientific names of the evaluated species must be written in italics.
• One reference needs to be corrected (as indicated in the PDF).
• The issue regarding the standard deviations mentioned in the PDF comments must be addressed.

Comments for author File: Comments.pdf

Author Response

Please find attached the revised version of the manuscript. The following edits have been completed as requested:

1. All scientific terms (e.g., Escherichia coli, in vitro, Streptococcus mitis) have been properly italicized throughout the text.

2. The previous references, including Ahmed et al., have been replaced with more recent and appropriate citations. All updated references are highlighted in yellow for your convenience.

3. The standard deviation (SD) comment under the last table has been reviewed and adjusted to reflect the correct statistical interpretation.

Please let me know if there are any further modifications needed or if you'd like me to proceed with the next steps.

Respectfully,

Doaa Ali

Author Response File: Author Response.docx