Harnessing Copper Nanoparticles for Antimicrobial Applications: Advances and Challenges

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript "Harnessing Copper Nanoparticles for Antimicrobial Applications: Advances and Challenges" gives a concise, up-to-date, and organized review of the function of CuNPs to mitigate AMR. There is a lot of scientific and therapeutic weight to the subject, and the manuscript does a good job of laying out the pros and downsides of using CuNPs. We value the cytotoxicity argument, the historical context, and the mechanistic insights. However, there is a lack of  some clarity and scientific rigor, deeper critical analysis, those should be addressed before going to acceptance.

 

1. It would be better to introduce with the Overview of the global challenge of antimicrobial resistance and the need for alternative searches. And then what this manuscript intends. It will bring the systematically organization of the plot…..
2. Although the authors mentioned here a comprehensive systemic review, but it lacks any search databases or keywords used.
3. Inclusion of the reason behind the drug resistance and the demand of use of nanoparticles as an alternatives in this context can improve the clarity of introduction.
4. Page 2, line no. 63, remove extra bracket…“……..livestock production [4])”.
5. Input the reference within the sentence….Page 3, line no. 122-123 …”especially after the 122 COVID-19 pandemic. [21]”
6. Use fullstop at the end of the sentence….Page 3, line no. 129 “….hospital 128 surfaces, and textiles [22]”, Page 6, line no. 251…”susceptible to oxidation without a protective coating [76], [77]”, Page 15, line no. 577…”and accelerated epithelializa-577 tion and collagen deposition in vivo [143]”
7. Write in italics of the scientific name of the microorganisms…..page 5, line no. 175…..”showed strong antimicrobial activity against Pseudomonas aeruginosa”; Page 9, line no. 329….”CuNPs interact with ergoste-329 rol-rich membranes, as shown in Candida tropicalis” Page 9, line no. 355 “caused oxidative lesions in the DNA of Klebsiella pneumoniae”
8. Authors does not mentioned the dose dependend toxicity threshold of CuNP in the antimicrobial section.
9. A comparative strengths and limitations of Cu and other NP in tabular form can enhance the understanding of this review.
10. Antimicrobial process (reactive oxygen species (ROS) production, membrane rupture, and Cu²⁺ ion release) appear more than once. To minimize on repetition, think about using cross-referencing or condensing.
11. Rewrite the sentence… page 9 …”Morphology plays a crucial role: in S. aureus, copper nanorods with sharp ends 334 showed up to 3.4-fold higher efficacy in membrane rupture than spherical nanoparticles 335 [72].”
12. Please correct the name, page 9, line no. 341…. “Catheters coated with 341 CuNP suppressed Candida aureus biofilms by 89%,”, Candida aureus? Or, Candida auris? Which is right?...
13. Please correct “as demonstrated by 389 [NO_PRINTED_FORM] [107]” page 10
14. In section 4.1, authors says the difference of mechanism of action between gram-positive and gram-negative bacteria is structural features. Is there only structural differences responsible for this? …..and image showing no peptidoglycan layer in case of gram-negative bacteria……modify it
15. Improve the resolution of figure 4.
16. Figure 4 and Figure 5 are lack detailed captions explaining mechanisms/processes. Each figure need self-explanatory, with key elements labeled clearly.
17. Use proper reference style , page 18, line no. 697…”Stimuli-responsive CuNPs that are activated only under acidic 697 tumor environment or by external irradiation have also shown promise as a way to mini-698 mize collateral tissue damage (Gong et al., 2025).”
18. The majority of manuscript summarizes prior research without providing adequate critical analysis of contradictory results, dose-dependent differences, or limitations in performance. Include this

Author Response

 

Comments 1: [Page 3, line no. 100....”I is important to mention that the nanoparticles can not only act independently, but also in synergy with antibiotics, improving drug efficacy, and disrupting resistance mechanisms, such as biofilm formation [14].”..... it will be “It” instead of “I”.....please correct]

Response 1: We appreciate the reviewer for carefully pointing out this typographical error. The correction has been made by replacing “I” with “It” on page 3, line 100. The sentence now correctly reads:
“It is important to mention that the nanoparticles can not only act independently, but also in synergy with antibiotics, improving drug efficacy, and disrupting resistance mechanisms, such as biofilm formation [14].”

Comments 2: [It would be better if the authors include those searching methods and used keywords in this manuscript.]
Response 2: We thank the reviewer for this valuable suggestion. The section describing the

search methodology has been included, specifically, the databases consulted, the search period, and the keywords employed in the literature search were added in the Introduction section (page 2, lines 55–63).

Comments 3: [In Figure 3, still it is not showing no peptidoglycan layer in case of gram-negative bacteria....please correct this]
Response 3: We appreciate the reviewer’s attentive observation. The figure has been corrected accordingly. A thin peptidoglycan layer has now been clearly added to the schematic representation of gram-negative bacteria in Figure 3 (page 13, line 520) to accurately reflect their structural characteristics.

It is important to mention that all the manuscript was revised by a native English speaker, that is a co-author of this document (Keith D. L. Lira). Thank you very much for all the comments.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

In this manuscript, Diogo S. Pellosi and colleagues review advances in CuNP synthesis and its applications in antimicrobial applications. They further summarize CuNP applications in medical devices, wound dressings, textiles, and packaging, as well as their potential toxicity and impacts. However, the authors should further address the following issues before considering accepting the manuscript.

 

1. For Figure 4, Biomedical application of copper oxide nanoparticles (modified by author). The authors merely deleted the word “oxide” from Figure 4 itself. The original figure was titled “Biomedical Application of Copper Oxide Nanoparticles.” Why did they fail to cite the source? This is evidently the same figure, with only the word “oxide” removed, yet they retained the original caption referring to “copper oxide nanoparticles.” Notably, copper nanoparticles consist of metallic copper, whereas copper oxide nanoparticles are already oxidized and thus generally more chemically stable. There should be some differences between the two types of nanosystems. Please be scientific.
2. Figure 1 shows the different approaches for copper nanoparticle synthesis. Why is there no clear labeling of the laser ablation process in the figure?
3. As depicted in Section 2.3 Biological (Green) Synthesis, there are diverse methods for green synthesis. Actinomycetes widely recognize the generation and various uses of nanoparticles. It also plays an important role in synthesis. Can you add and compare it?
4. This review paper also compares the recent advances of copper nanoparticles; some of the other advanced synthesis methods can be mentioned a little bit within the comparison section (e.g., Green microwave-assisted synthesis, microfluidic synthesis, ultrasound-assisted chemical reduction, photochemical synthesis).
5. For the application section, why do the authors not include the clinical translation or current translated patents for the copper nanoparticles?
6. What is the copper homeostasis in physiology and cancer?
7. It would be better if the authors could compare other metal nanoparticles with copper nanoparticles to highlight their application.
8. Please list clearly the software or platform used for the review paper.

Author Response

 

In this manuscript, Diogo S. Pellosi and colleagues review advances in CuNP synthesis and its applications in antimicrobial applications. They further summarize CuNP applications in medical devices, wound dressings, textiles, and packaging, as well as their potential toxicity and impacts. However, the authors should further address the following issues before considering accepting the manuscript.   Comments 1: [For Figure 4, Biomedical application of copper oxide nanoparticles (modified by author). The authors merely deleted the word “oxide” from Figure 4 itself. The original figure was titled “Biomedical Application of Copper Oxide Nanoparticles.” Why did they fail to cite the source? This is evidently the same figure, with only the word “oxide” removed, yet they retained the original caption referring to “copper oxide nanoparticles.” Notably, copper nanoparticles consist of metallic copper, whereas copper oxide nanoparticles are already oxidized and thus generally more chemically stable. There should be some differences between the two types of nanosystems. Please be scientific.]

Response 1: Thank you for this feedback. Figure 4 is indeed the same as the one presented in the original article. [It was modified by the authors, as indicated in the corresponding comment. However, since copper nanoparticles and copper oxide nanoparticles share many similar applications (albeit through different mechanisms) there are certain cases where their applications do not fully overlap. Therefore, a new figure has been created to illustrate the most common and emerging biomedical applications of metallic copper nanoparticles.]

Comments 2: [Figure 1 shows the different approaches for copper nanoparticle synthesis. Why is there no clear labeling of the laser ablation process in the figure?]

Response 2: Thank you for this remark. The laser ablation method wass identified, in the image, as a laser beam inside the circle regarding “physical processes” in figure 1.   Comments 3: [As depicted in Section 2.3 Biological (Green) Synthesis, there are diverse methods for green synthesis. Actinomycetes widely recognize the generation and various uses of nanoparticles. It also plays an important role in synthesis. Can you add and compare it?] Response 3: Thank you for pointing this out. We agree with this comment, so [we have changed this in section 2.3, page 7, lines 280-289. “[...] it depends of the source and type of plant extract, as well as the concentration of phytochemicals, in addition to the extraction method, which influences the characteristics of a nanostructure, potentially resulting in particles with less uniform size distribution and morphology [66] [63]. On the other hand, the actinomycetes are widely recognized for the generation and various uses of nanoparticles, their composition, with greater enzymatic diversity, additional bioactives, which makes them more efficient than other bacteria, especially to improve control over the shape and size of nanoparticles and their application on a large scale [10.5829/idosi.aejsr.2016.11.3.22798], [10.1007/s13204-014-0304-7] [10.1016/j.apjtb.2015.04.007], [10.1007/s13205-017-0930-3].”]   Comments 4: [This review paper also compares the recent advances of copper nanoparticles; some of the other advanced synthesis methods can be mentioned a little bit within the comparison section (e.g., Green microwave-assisted synthesis, microfluidic synthesis, ultrasound-assisted chemical reduction, photochemical synthesis).] Response 4: We appreciate this valuable suggestion. [The mentioned methods—microwave-assisted green synthesis, microfluidic synthesis, ultrasound-assisted reduction, and photochemical approaches—have been incorporated into the manuscript (page 7, Section 2.4 “Comparison of synthesis methods regarding particle size, morphology, and stability”). Additionally, other recently developed synthesis strategies, such as solvothermal, hydrothermal, electrochemical, and plasma-assisted methods, have also been included to provide a more comprehensive overview of advanced CuNP synthesis approaches.]   Comments 5: [For the application section, why do the authors not include the clinical translation or current translated patents for the copper nanoparticles?] Response 5: Thank you for this observation. [A table has been included in section 5. “Applications in biomedical field” (Page 20) listing the most recent patents regarding the use of copper nanoparticles in the clinical field.]   Comments 6: [What is the copper homeostasis in physiology and cancer?] Response 6: Thank you for pointing that out. [The following text was added to section 6.1, page 22 paragraph two, lines 850 - 860: In humans, copper functions as a cofactor for enzymes fundamental to metabolism, such as cytochrome C, important in cellular respiration, and superoxide dismutase (SOD), an antioxidant enzyme. Absorbed mainly by the duodenum, copper is transported through the blood associated with chaperones to target organs, and can be stored in the liver. When in excess, copper is mainly released in feces and bile secretion. In cancer, Cu can act as a modulator in cell signaling, stimulating cell proliferation, angiogenesis, and metastasis, both at the transcriptional and substrate levels. In general, cancer cells have disordered copper regulation with higher demands for the metal than a healthy cell. Therapies involving copper complexes stimulate an increase in intracellular copper concentration in order to cause their cytotoxic effects and decrease its availability in circulation.”]   Comments 7: [It would be better if the authors could compare other metal nanoparticles with copper nanoparticles to highlight their application.] Response 7: Thank you for your valuable suggestion. [We added a table 2 in 2.5 section with comparison of copper nanoparticles and other metallic nanoparticles as suggested by another reviewer and a paragraph highlighting the main advantages and limitations of copper and also other metallic nanoparticles has been added on page 8-9.]   Comments 8: [Please list clearly the software or platform used for the review paper.] Response 8: Thank you for bringing this to our attention. The Reviewer 1 also asked the same to us. We used Google Scholar, Web of Science, Science Direct and PubMed platforms.

 

4. Response to Comments on the Quality of English Language

Point 1: (x) The English is fine and does not require any improvement.

Response 1: Thank you for your comment.

5. Additional clarifications

All of the suggestions and comments could improve a lot the manuscript. Thank you for the revision.

 

 

Reviewer 3 Report

Comments and Suggestions for Authors

Dear authors,

The review paper is well written, informative, and clearly organized. It provides significant information on AMR, Cu, synthesis methods of CuNPs, and their antimicrobial applications. However, I have a few minor comments and suggestions to further improve the manuscript. 

1. Give the latest data (2024 or 2025) on AMR to provide up-to-date information
2. Authors should provide the reference number in Table 1.
3. Authors can add details on how the different morphologies of NPs influence the antimicrobial activity
4. Include top-down and bottom-up approaches.
5. Algal-mediated synthesis of CuNPs is missing. Add these details to the biological (green) synthesis.
6. Authors can further improve their manuscript by including advanced concepts such as quorum sensing and quorum quenching.

Author Response

Dear authors,

 

The review paper is well written, informative, and clearly organized. It provides significant information on AMR, Cu, synthesis methods of CuNPs, and their antimicrobial applications. However, I have a few minor comments and suggestions to further improve the manuscript.

 

Comments 1: [Give the latest data (2024 or 2025) on AMR to provide up-to-date information]

Response 1: We appreciate the suggestion, so [we added more current references on the topic as requested. Was added that sentence on page 2, lines 65-68: “In the current scenario, projections indicate that between 2025 and 2050, the number of deaths associated with AMR will reach around 169 million. This figure highlights the urgency of developing new approaches to overcome this situation”].

Comments 2: [Authors should provide the reference number in Table 1.]

Response 2: Thank you for bringing this to our attention. We have fixed the mistake.   Comments 3: [Authors can add details on how the different morphologies of NPs influence the antimicrobial activity]. Response 3: We appreciate the suggestion, so [we added two paragraphs on page 13, in the session 4.4, lines 518-531, describing how the different morphologies influence the antimicrobial activity, including ROS generation, physical action and ionic release. The paragraphs included are  “This is noted because of different morphologies …” and “Shapes with points or edges …”].   Comments 4: [Include top-down and bottom-up approaches.] Response 4: Thank you for your suggestion. [A paragraph describing the approaches bottom up and top down was added in a 2. section about Synthesis of Copper nanoparticles, on page 4, lines 178-187. “Nanomaterials can be obtained from two main approaches: top-down and bottom-up. The top-down approach means obtaining the nanomaterials by breaking down a larger portion of the material to obtain the desired nanostructure (from macro to nano). This is generally used in the areas of microelectronics, engineering, and physics, and usually involves physical methods. The bottom-up approach implies the components of the material, initially present at the atomic scale, self-organize into nanostructured materials, that is, nanostructures are built atom by atom, or molecule by molecule, or through clusters of atoms or molecules. This is commonly used in chemistry and biology, and chemical methods are generally more employed”].   Comments 5: [Algal-mediated synthesis of CuNPs is missing. Add these details to the biological (green) synthesis.] Response 5: Thank you for your suggestion. [A paragraph on the CuNPs mediated by algae has been added on page 7, lines 298-307.: “There is also great interest in the synthesis process using algae. Their composition, including proteins and secondary metabolites, allows them to function as nanobiofactories, hyperaccumulating metals and transforming them into nanoparticles. Additionally, their good cost-effectiveness and the possibility of large-scale application in nanoparticles of different metals stand out. This method is related to specific released pigments and, in the case of copper nanoparticles, brown algae are most commonly used. Like other green methods, the low or non-existent toxicity allows the synthesized nanoparticles to be used in the biomedical field more safely [https://doi.org/10.3390/biom10111498] [https://doi.org/10.1016/j.btre.2014.12.001]. [https://doi.org/10.3109/07388551.2010.550568].”].   Comments 6: [Authors can further improve their manuscript by including advanced concepts such as quorum sensing and quorum quenching.] Response 6: This feedback was essential, so [we appreciated it and added a new section called 4.5 Copper-based Nanoparticles in the Inhibition of Quorum Sensing on page 16 of review.]

 

4. Response to Comments on the Quality of English Language

Point 1: (x) The English is fine and does not require any improvement.

Response 1: Thank you for your comment.

5. Additional clarifications

All of the suggestions and comments could improve a lot the manuscript. Thank you for the revision.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

 

Authors addressed most of the comments satisfactorily, but still few points need to address.

1. Page 3, line no. 100….”I is important to mention that the nanoparticles can not only act independently, but also in synergy with antibiotics, improving drug efficacy, and disrupting resistance mechanisms, such as biofilm formation [14].”….. it will be “It” instead of “I”…..please correct
2. It would be better if the authors include those searching methods and used keywords in this manuscript.
3. In Figure 3, still it is not showing no peptidoglycan layer in case of gram-negative bacteria….please correct this

Author Response

Comments 1: [Page 3, line no. 100....”I is important to mention that the nanoparticles can not only act independently, but also in synergy with antibiotics, improving drug efficacy, and disrupting resistance mechanisms, such as biofilm formation [14].”..... it will be “It” instead of “I”.....please correct]

Response 1: We appreciate the reviewer for carefully pointing out this typographical error. The correction has been made by replacing “I” with “It” on page 3, line 100. The sentence now correctly reads:
“It is important to mention that the nanoparticles can not only act independently, but also in synergy with antibiotics, improving drug efficacy, and disrupting resistance mechanisms, such as biofilm formation [14].”

Comments 2: [It would be better if the authors include those searching methods and used keywords in this manuscript.]
Response 2: We thank the reviewer for this valuable suggestion. The section describing the

search methodology has been included, specifically, the databases consulted, the search period, and the keywords employed in the literature search were added in the Introduction section (page 2, lines 55–63).

Comments 3: [In Figure 3, still it is not showing no peptidoglycan layer in case of gram-negative bacteria....please correct this]
Response 3: We appreciate the reviewer’s attentive observation. The figure has been corrected accordingly. A thin peptidoglycan layer has now been clearly added to the schematic representation of gram-negative bacteria in Figure 3 (page 13, line 520) to accurately reflect their structural characteristics.

It is important to mention that all the manuscript was revised by a native English speaker, that is a co-author of this document (Keith D. L. Lira). Thank you very much for all the comments.

Reviewer 2 Report

Comments and Suggestions for Authors

I have reviewed the revised manuscript, including the authors’ responses to the reviewers, and they have addressed my comments.

Author Response

Thank you for your feedback