Evaluation of Superparamagnetic Fe₃O₄-Ag Decorated Nanoparticles: Cytotoxicity Studies in Human Fibroblasts (HFF-1) and Breast Cancer Cells (MCF-7)

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Title: “Evaluation of Superparamagnetic Fe3O4-Ag Decorated Nanoparticles: Cytotoxicity Studies in Human Fibroblasts (HFF-1) and Breast Cancer Cells (MCF-7)”

 Brief Summary:

This scientific study evaluates the cytotoxicity of superparamagnetic Fe3O4-Ag decorated nanoparticles in human fibroblasts (HFF-1) and breast cancer cells (MCF-7). The study's primary goal is to understand the potential for therapeutic application as well as evaluating the safety profile of these nanoparticles for therapeutic applications. The researchers conducted a series of in vitro experiments to determine the cytotoxic effects, cellular uptake, and mechanisms of cell death induced by the nanoparticles.

 

Main findings included:

- Fe3O4-Ag nanoparticles exhibited minimal cytotoxicity in MCF-7 breast cancer cells, with minimal dose-dependent decrease in cell viability.

- In contrast, the nanoparticles demonstrated minimal cytotoxicity in HFF-1 human fibroblasts, suggesting a selective effect on cancer cells.

- Cellular uptake studies revealed that MCF-7 cells internalized a higher concentration of nanoparticles compared to HFF-1 cells.

-Studies indicated that the cytotoxic effect in both HFF-1 and MCF-7 cells was primarily due to the involvement of Reactive Oxygen Species (ROS), but due to lack of specificality the nanoparticles could also cause harm to non-cancerous viable cells.

 

Specific Comments:

1. P.4 line 12-15 (Introduction):

   a. The rationale for selecting Fe3O4-Ag nanoparticles for this study is not clearly stated.

      i. Provide a brief explanation of why these specific nanoparticles were chosen, highlighting their unique properties and potential advantages in cancer therapy. Explanation of formation is clear but selection criteria for research was unclear.

 

2. P.5 line 18-20 (Materials and Methods):

   a. The specific concentrations of nanoparticles used in the cytotoxicity assays are not specified in regard to the parameters that were chosen for this research.

      i. Include the specific concentrations tested to allow for reproducibility with an explanation of why it was chosen along with comparison/references from other studies. The sentence reports that “different concentrations of nanoparticles from 0.1 to 100…” without further explanation or detail.

 

3. P.6 line 5-7 (Cell Culture):

   a. The culture conditions of HFF-1 and MCF-7 were not listed.

      i. Indicate the supplier of the cell lines and the conditions at which samples were obtained, retrieved, and stored to ensure the author delivers credibility to the source as well as allowing for reproducibility.

 

4. P.7 lines 10-12 (Cytotoxicity Assay):

   a. The method of measuring cell viability (e.g., MTT assay, trypan blue exclusion) is not described in detail.

      i. Provide a comprehensive description of the cytotoxicity assay procedure to ensure clarity and reproducibility.

Author Response

RESPONSE TO REVIEWER 1

 

Comments and Suggestions for Authors

Title: “Evaluation of Superparamagnetic Fe3O4-Ag Decorated Nanoparticles: Cytotoxicity Studies in Human Fibroblasts (HFF-1) and Breast Cancer Cells (MCF-7)”

 Brief Summary:

This scientific study evaluates the cytotoxicity of superparamagnetic Fe3O4-Ag decorated nanoparticles in human fibroblasts (HFF-1) and breast cancer cells (MCF-7). The study's primary goal is to understand the potential for therapeutic application as well as evaluating the safety profile of these nanoparticles for therapeutic applications. The researchers conducted a series of in vitro experiments to determine the cytotoxic effects, cellular uptake, and mechanisms of cell death induced by the nanoparticles.

 

Main findings included:

- Fe3O4-Ag nanoparticles exhibited minimal cytotoxicity in MCF-7 breast cancer cells, with minimal dose-dependent decrease in cell viability.

- In contrast, the nanoparticles demonstrated minimal cytotoxicity in HFF-1 human fibroblasts, suggesting a selective effect on cancer cells.

- Cellular uptake studies revealed that MCF-7 cells internalized a higher concentration of nanoparticles compared to HFF-1 cells.

-Studies indicated that the cytotoxic effect in both HFF-1 and MCF-7 cells was primarily due to the involvement of Reactive Oxygen Species (ROS), but due to lack of specificality the nanoparticles could also cause harm to non-cancerous viable cells.

 

Specific Comments:

1. P.4 line 12-15 (Introduction):
2. The rationale for selecting Fe3O4-Ag nanoparticles for this study is not clearly stated.

RESPONSE:

This was addressed and clarified within the new version of the manuscript, the changes and additions of text were marked in the text with color.

1. Provide a brief explanation of why these specific nanoparticles were chosen, highlighting their unique properties and potential advantages in cancer therapy. Explanation of formation is clear but selection criteria for research was unclear.

RESPONSE:

The Fe₃O₄-Ag nanoparticles were selected for evaluation in this study due to their extensive use in various biomedical applications, including antibacterial and antifungal treatments. As part of the research development, we considered it important to start from the working hypothesis that it would be necessary to evaluate the cytotoxicity of these Fe₃O₄-Ag particles. While previous studies have extensively evaluated the physicochemical properties of these magnetoplasmonic nanoparticles, this study focuses on assessing their cytotoxic properties. We believe that investigating cytotoxicity is beneficial and can be of great value to the field, providing a comprehensive understanding of the potential benefits and unique properties of Fe₃O₄-Ag nanoparticles in cancer therapy.

 

2. P.5 line 18-20 (Materials and Methods):
3. The specific concentrations of nanoparticles used in the cytotoxicity assays are not specified in regard to the parameters that were chosen for this research.

RESPONSE:

The reviewer is correct. To improve understanding of the manuscript, the concentrations used, and the reasons for this are described in the methodology. The text can now be read as follows in Methodology section 2.3.1.:

“The concentrations of nanoparticles used in this work for all the bioassays were selected according to the suggestions issued by the OECD through the Nanomaterials Series on the Safety of Manufactured Nanomaterials No. 85 described in the technical brochure for the Evaluation of in vitro methods for human hazard assessment applied in the OECD Testing Program for the Safety of Manufactured.”

1. Include the specific concentrations tested to allow for reproducibility with an explanation of why it was chosen along with comparison/references from other studies. The sentence reports that “different concentrations of nanoparticles from 0.1 to 100…” without further explanation or detail.

RESPONSE

We agree with the reviewer's observation. To enhance the understanding of the methodology described in our article, we have included the point concentrations of the nanoparticles evaluated here. This information can be found in the Materials and Methods section 2.3.2 as follows:

“The effect of Fe₃O₄ and Fe₃O₄-Ag decorated nanoparticles on cell viability was evaluated using the MTT reduction, following the recommendations provided by the OECD testing programme for the safety of manufactured nanomaterials. Thus, MCF-7 and HFF-1 cells were seeded at a density of 10,000 cells per well in a 96-well plate and exposed to different concentrations of nanoparticles from 0.1, 1, 5,10, 25, 50, and  100 µg/mL.”

3. P.6 line 5-7 (Cell Culture):
4. The culture conditions of HFF-1 and MCF-7 were not listed.
5. Indicate the supplier of the cell lines and the conditions at which samples were obtained, retrieved, and stored to ensure the author delivers credibility to the source as well as allowing for reproducibility.

RESPONSE:

In response to the reviewer's comment, we have included details on the specifications and suppliers of the cell lines, along with a description of their cultivation and maintenance in section 2.3.1 on cell culture. Our research group frequently uses cell culture for nanotoxicological evaluation to assess various nanomaterials. Therefore, we have included references to support the methodology employed in this study.

The additions in the text can be found in section 2.3.1. as follows:

“The human breast adenocarcinoma MCF-7 cells (ATCC-HTB-22) and Human Fibroblasts HFF-1 (SRC-1041) were purchased from the American Type Culture Collection (ATCC, Manasas, Virginia, USA). The cell lines were thawed, cultivated, and maintained following the provider’s instructions.  Cell lines were grown in Petri dishes for cell culture at a temperature of 37◦C in 5% CO₂ atmosphere in Dulbecco’s Modified Eagle’s Medium (DMEM), supplemented with 10% Fetal Bovine Serum (FBS, BenchMark, Geminis Bio Products), 1% Penicillin-Streptomycin (Sigma-Aldrich), 1% L-glutamine, and 2 g/L of sodium bicarbonate, as previously described [1–4]

4. P.7 lines 10-12 (Cytotoxicity Assay):
5. The method of measuring cell viability (e.g., MTT assay, trypan blue exclusion) is not described in detail.
6. Provide a comprehensive description of the cytotoxicity assay procedure to ensure clarity and reproducibility.

RESPONSE:

We appreciate the reviewer's observation of improving the materials and methods section. We have added a more detailed description of the MTT depletion cytotoxicity assay methodology. This can be found in section 2.3.2. Cytotoxicity assay by MTT reduction, as follows:

 

“The effect of Fe₃O₄ and Fe₃O₄-Ag decorated nanoparticles on cell viability was evaluated using the MTT reduction, following the recommendations provided by the OECD testing programme for the safety of manufactured nanomaterials. The cytotoxicity effect of Fe₃O₄ and Fe₃O₄-Ag decorated nanoparticles was determined using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, a widely used method to assess cell viability[5,6]. HFF-1 and MCF-7 cells were seeded at a density of 10,000 cells per well in a 96-well plate and incubated in 100 μL of DMEM-supplemented media at 37°C in a 5% CO₂ atmosphere for 24 h. After this, Fe₃O₄ and Fe₃O₄-Ag decorated NPs were suspended in MiliQ-water at 1 mg/mL concentrations. Before each assay, each nanoparticle suspension was prepared freshly and underwent 10 minutes of sonication. Then, solutions of nanoparticles at different concentrations were made in DMEM media and promptly added to the cell cultures as described below.HFF-1 and MCF-7 cells were exposed to Fe₃O₄ and Fe₃O₄-Ag decorated NPs at 0.1; 1, 5, 10, 25, 50, and 100 μg/mL in a final volume of 100 μL of DMEM-supplemented media in a 96-well plate. Cells were incubated for 24 h at 37°C in a 5% CO₂ atmosphere using DMEM media. Afterward, the media was discarded, and cells were rinsed thrice with 200 μL of PBS 1x. Then, 10 μL of MTT and 90 μL of DMEM media were added to each well and incubated for 4 h at 37°C in a 5% CO₂ atmosphere for 24 h. After this, 100 μL of isopropanol was added to each well to dissolve formazan crystals, and the 96-well plate was incubated for 30 min at 25 °C in darkness. Absorbance readings were taken at 570 and 690 nm wavelengths using a microplate reader (ThermoFisher Scientific, Waltham, Massachusetts, USA). Each cytotoxicity experiment included a control group with a culture medium lacking NPs to establish a baseline. The positive control for inducing cell death consisted of cells treated with 1% (v/v) Triton X-100, while the negative control contained only cell culture medium without nanoparticles. These controls facilitated the comparison of cell viability between the negative and positive controls and the damage caused by various concentrations of NPs. Experiments were conducted in triplicate. Cell viability was calculated relative to the absorbance values of the positive control. “

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Reviewer Feedback to the Author:

The experimental studies need to be more systematic and scientifically robust to support the claims made. This will attract more attention from readers, as the current manuscript lacks this rigor.

Comments for author File: Comments.pdf

Author Response

RESPONSE TO REVIEWER 2:

 

Reviewer Feedback to the Author:

This research article effectively discusses the application of Superparamagnetic Fe3O4-Ag Decorated Nanoparticles and their cytotoxicity studies in Human Fibroblasts (HFF-1) and Breast Cancer Cells (MCF-7). It highlights the promising biocompatibility and low toxicity of Fe3O4 and Fe3O4-Ag nanoparticles at concentrations below 25 μg/mL, suggesting potential suitability for various biomedical applications. However, additional information is needed to strengthen this manuscript:

1. Hydroxyl Radicals Confirmation: Have you performed any electron paramagnetic resonance (EPR) studies to confirm the generation of reactive oxygen species (ROS)? Please provide experimental evidence to support that your proposed nanomaterials, Superparamagnetic Fe3O4-Ag Decorated Nanoparticles, can successfully generate ROS.

 

RESPONSE:

We appreciate the reviewer for providing suggestions to enhance the clarity of our work. We concur with the reviewer's perspective regarding the importance of identifying the chemical nature of the ROS induced by Fe₃O₄ and decorated Fe₃O₄-Ag in cells. However, in our experiments aimed at assessing potential ROS overproduction following exposure to NPs, we did not observe statistically significant changes. This suggests no discernible difference between the endogenous ROS levels and those induced by NPs in cells. This lack of statistical significance was consistent across most concentrations of both Fe3O4 and Fe3O4-Ag decorated NPs, where the ROS production did not differ significantly from that observed in the control group, where cells were cultured in medium without NPs, thereby producing endogenous ROS. Consequently, the experiments presented in Figure 7, comparing ROS levels induced in HFF-1 and MCF-7 cells exposed to both types of NPs, demonstrate that NPs in these cell models induced no statistically significant ROS overproduction.  One mechanism of action in future applications involving these NPs in combination with anticancer drugs could involve ROS overproduction. Therefore, as suggested by the reviewer, it would be beneficial to evaluate the specific types of ROS generated using techniques such as EPR. Additionally, complementary methods such as flow cytometry and confocal microscopy with specific kits can be employed to identify the primary radicals formed within the cells.

 

2. Zeta Potential Measurements: Conduct zeta potential measurements at a fixed pH and explain the interaction of your proposed nanomaterials. This information is crucial for understanding this research article.

 

RESPONSE:

Zeta potential measurements were conducted at a fixed pH to understand the interaction of the proposed nanomaterials. Figure 9 includes a graph showing the zeta potential as a function of pH for the Fe3O4 magnetic nanoparticles. Additionally, a discussion has been included to explain the interaction of these nanoparticles with the studied cells. This information is crucial for understanding the stability and behavior of the nanoparticles in the colloidal medium, as well as their potential cytotoxicity and biological interactions.

 

3. Scale Bar Improvement: In Figures 1 and 2 (pages 6 and 7), improve the scale bar, as it is currently difficult to read. Major Drawbacks of the Research Article:

RESPONSE:

In Figures 1 and 2 (pages 6 and 7), the scale bars have been improved, making the images clearer and more precise to read. The scale bars were enhanced, and the readability of the images is now clearer and more accurate.

 

4. The study is limited to in vitro evaluation of cytotoxicity and biocompatibility without assessing potential toxicity in animal models or clinical settings.

RESPONSE:

The reviewer's observation is indeed pertinent. This study aims to conduct an in vitro nanotoxicological assessment to determine whether exposure of cells to varying concentrations of Fe₃O₄ and Fe₃O₄-Ag decorated NPs induces cytotoxic effects. Our investigation, encompassing four distinct assays—MTT viability tests, ROS production, hemocompatibility, and nitrite production (as an indicator of in vitro inflammation)—revealed that at the majority of NP concentrations tested, there was no significant cytotoxicity or oxidative stress induction observed. However, concentrations exceeding 50 ug/mL elicited apparent hemolytic and inflammatory effects in vitro. Consequently, based on these findings, it can be established that NP concentrations ranging from 0.1 to 25 ug/mL exhibit biocompatibility. Such preclinical trials are crucial for ensuring the safe utilization of NPs and identifying effective concentration ranges where desired effects can be pursued in other biological models, such as future applications in delivering antineoplastic drugs. Therefore, before advancing to in vivo trials, additional nanotoxicological investigations in various cancer cell lines will be necessary to ascertain whether the observed effects align with expectations and could potentially be employed as anti-proliferative nanosystems.

5. The article lacks a comprehensive discussion on the limitations of the study, such as using only two cell lines and the absence of comparisons with other nanoparticle systems.

RESPONSE:

Following the recommendation made by the reviewer, we added a short discussion which alludes to this comment. The text can be found in the cytotoxicity assay results section:

“As observed in Figure 6, the cell viability on HFF-1 fibroblasts changed across the different concentrations of Fe₃O₄ and Fe₃O₄-Ag decorated nanoparticles. However, none of them significantly reduced cell viability; thus, all the concentrations tested were not cytotoxic. On the other hand, the cell viability of MCF-7 breast cancer cells did not change upon the exposure of cells to different concentrations of nanoparticles, indicating that all of them are compatible and allowed the cells to grow as in the control. These results indicate that Fe₃O₄ and Fe₃O₄-Ag nanoparticles are not cytotoxic. However, it is essential to ensure that these nanoparticles do not exhibit undesirable cytotoxicity towards normal non-cancerous cells, as this could limit their viability and safety for clinical use[1,2]. Unlike our study, which focused on comparing the cytotoxic effects of Fe3O4 and Fe3O4@Ag NPs, other studies have evaluated similar nanosystems conjugated with thiolated chitosan in different cell lines such as HFF-1 fibroblasts, MC3T3-E1 osteoblasts, and the osteosarcoma cell line MG63. These studies used higher concentrations ranging from 5 to 200 ug/mL. Specifically, in HFF-1 cells, a 50% decrease in cell viability was observed starting from 100 µg/mL[3] Careful evaluation of the effects of nanoparticles on cancerous and non-cancerous cell lines at various concentrations is essential to achieve the appropriate balance between therapeutic efficacy and biocompatibility[4].

 

6. The article does not provide a detailed analysis of the mechanisms underlying the differential response of normal and cancer cells to the nanoparticles in terms of ROS production and cytotoxicity.

RESPONSE:

We thank the reviewer for this observation. Our findings from the cytotoxicity and ROS production assays indicate that there were no cytotoxic effects observed in HFF-1 fibroblast cells or MCF-7 breast cancer cells when exposed to either type of NPs across all concentrations tested. Regarding ROS production, we observed a lower induction of ROS in HFF-1 cells only at the concentration of 100 ug/mL with Fe3O4-Ag decorated NPs; however, no statistically significant differences were observed at other concentrations in either cell line. Consequently, we conclude that both types of NPs exhibited similar effects on both cell lines regarding toxicity and oxidative stress. Therefore, there is no discernible differential effect between the cell lines, suggesting a consistent mechanism of action across both.

A feasible explanation regarding this can be found in the manuscript as follows:

“As observed in Figure 6, the cell viability on HFF-1 fibroblasts changed across the different concentrations of Fe₃O₄ and Fe₃O₄-Ag decorated nanoparticles. However, none of them significantly reduced cell viability; thus, all the concentrations tested were not cytotoxic. On the other hand, the cell viability of MCF-7 breast cancer cells did not change upon the exposure of cells to different concentrations of nanoparticles, indicating that all of them are compatible and allowed the cells to grow as in the control. These results indicate that Fe₃O₄ and Fe₃O₄-Ag nanoparticles are not cytotoxic. However, it is essential to ensure that these nanoparticles do not exhibit undesirable cytotoxicity towards normal non-cancerous cells, as this could limit their viability and safety for clinical use[1,2].”

 

This revision aims to clarify the findings and improve the structure and coherence of the text.

Suggestions for Improving the Research Article:

7. Discuss Limitations: Address the limitations like the use of only two cell lines (HFF-1 and MCF-7) and the lack of comparison with other nanoparticle systems to provide a clearer scope and generalizability.

RESPONSE:

We appreciate the comment made by the reviewer, and to reinforce the proposed idea, we add the following text:

“The results showed that neither type of nanoparticle significantly reduced cell viability in HFF-1 fibroblasts, indicating that they are non-cytotoxic at the tested concentrations on a non-cancerous cell line. Similarly, MCF-7 breast cancer cells did not exhibit a significant change in viability when exposed to different nanoparticle concentrations, suggesting that these nanoparticles are biocompatible and allow normal cell growth. While evaluating the cytotoxic effects of these nanosystems in combination with other antineoplastic drugs across various cell lines from healthy tissues and neoplasms is crucial, this study primarily focuses on elucidating the biocompatibility of magnetite nanoparticles. This highlights the potential of Fe3O4 and Fe3O4-Ag nanoparticles for biomedical applications, given their compatibility with both normal and cancerous cells.”

 

8. Further Experiments: Investigate the mechanisms of differential response in normal vs. cancer cells regarding ROS production and cytotoxicity for therapeutic insights. For further understanding, the following research article might be helpful: "Covalent organic framework nanosheets as an enhancer for light-responsive oxidase-like nanozymes: Multifunctional applications in colorimetric sensing, antibiotic degradation, and antibacterial agents," ACS Sustainable Chemistry & Engineering, 2023, 11, 6956–6969.2

RESPONSE:

We appreciate the reviewer's recommendation and fully agree on the importance of delving deeper into the mechanisms underlying the toxicity of the NPs evaluated in this study. Your suggestion is invaluable for our future projects, which aim to explore the in vitro antiproliferative effects of Fe₃O₄-Ag decorated NPs across various cancer cell lines and compare their impact in cellular models derived from healthy tissues.

Likewise, the suggested reference was included in the manuscript.

65. Krishna Kumar, A.S.; Tseng, W.-B.; Arputharaj, E.; Huang, P.-J.; Tseng, W.-L.; Bajda, T. Covalent Organic Framework Nanosheets as an Enhancer for Light-Responsive Oxidase-like Nanozymes: Multifunctional Applications in Colorimetric Sensing, Antibiotic Degradation, and Antibacterial Agents. ACS Sustain Chem Eng 2023, 11, 6956–6969, doi:10.1021/acssuschemeng.2c07141.

 

9. Higher Concentrations: Evaluate the effects of nanoparticle concentrations above 100 μg/mL to understand toxicity and determine optimal ranges for biomedical use.

RESPONSE:

 

We appreciate the reviewer's suggestion and agree with the importance of evaluating higher concentrations of NPs. There is a precedent in the literature where similar NPs were tested at concentrations of 100, 150, and 200 µg/mL, revealing varying effects on HFF-1 and MC3T3-E1 cells compared to osteosarcoma cells. Our study confirms that the NPs we synthesized exhibit no cytotoxicity at concentrations up to 100 µg/mL[3]. However, before exploring biomedical use in vivo, some future applications within our group aim to explore the potential synergistic effects of these NPs with anticancer drugs. In such a case, it will be necessary to assess higher concentrations to demonstrate their cytotoxic effects on cancer cells and enhance their in vitro antiproliferative impact in cancer cell models.

 

Address these points to enhance the research relevance and contribution to the importance of nanoparticles for biomedical development.

RESPONSE:

In response to the reviewer's suggestions, we have emphasized the significance of our findings regarding the implications of NPs in future biomedical applications. This emphasis can be found in the manuscript with the following passage:

“Reactive oxygen species (ROS) production by Fe3O4 and Fe3O4-Ag nanoparticles was also examined to assess their biocompatibility in aims of their future therapeutic potential. The study found that higher concentrations of Fe3O4-Ag nanoparticles decreased ROS production in both HFF-1 and MCF-7 cells, while Fe3O4 nanoparticles were more effective in generating ROS at these concentrations. However, excessive ROS production can harm normal cells, underscoring the need to evaluate nanoparticle concentrations to balance therapeutic efficacy and safety carefully. These findings contribute to understanding nanoparticle interactions with cellular oxidative mechanisms, which are crucial for developing safe and effective nanoparticle-based therapies. Altogether, the results herein indicated that it is possible to use Fe3O4-Ag nanoparticles as future drug delivery agents and enhance the synergy of their physicochemical properties with the antiproliferative mechanisms of the anticancer agents, also seeking the repositioning of front-line drugs for the treatment of public health diseases.”

RESPONSE:

 

We have detected that the text that appears below does not correspond to our work, we consider that the work it refers to is the application of mesoporous silica

nanoparticles (MSNs) in biosensors for cancer biomarker detection, therefore we do not consider responding regarding that material that is not the one reported by us.

 

1. Summary and Strengths:

 This review article effectively discusses the application of mesoporous silica

nanoparticles (MSNs) in biosensors for cancer biomarker detection. Key points

covered include cancer biomarkers, MSNs in biosensors, ASSURED criteria, various

biosensor types, sensitivity and specificity, and molecularly imprinted polymer-silica

nanocomposites. The content is well-structured and organized.

2. Key Drawbacks:

 -Sensitivity and Specificity Challenges: While MSNs enhance biosensor

performance and sensitivity, the limits of detection (LOD) for early screening remain

critical. Specificity is essential to avoid false positives, especially since tumor markers

can be linked to various tumors.

◼ Complexity of Biomarker Detection: The article needs to address the

complexity involved in detecting biomarkers accurately.

◼ Integration and Commercialization: The manuscript should discuss the

integration of MSNs in commercial biosensors and the challenges faced.

◼ Regulatory Hurdles: Highlighting the regulatory challenges for bringing such

biosensors to market is necessary.

◼ Cost and Accessibility: Addressing the cost and accessibility issues will be

crucial for practical applications.

3. Suggestions for Improvement:

◼ Improving Sensitivity and Specificity: Provide strategies to enhance the

sensitivity and specificity of biosensors.

◼ Addressing Regulatory and Commercialization Challenges: Discuss the

pathways to overcoming regulatory and commercialization hurdles.

◼ Exploring Cost-Effective Fabrication and Integration: Suggest methods for

cost-effective fabrication and integration of MSNs in biosensors.

◼ Highlighting Emerging Trends and Future Directions: Identify emerging

trends and potential future directions in this field.

◼ Providing Comprehensive Evaluation of Limitations: Offer a thorough

evaluation of the current limitations and propose solutions.

4

4. Additional Information to Strengthen the Review:
5. In Vitro and In Vivo Studies: Include studies that show the efficacy of MSNs in

both in vitro and in vivo settings.

1. Comparative Analysis: Provide comparative analysis with other nanoparticlebased biosensors.

iii. Real-Time Detection and Point-of-Care Testing: Discuss the potential for real-time

detection and point-of-care applications.

1. Integration with Advanced Technologies: Explore the integration of MSNs with

advanced technologies like microfluidics and nanofluidics.

1. Mechanistic Studies: Include mechanistic studies to explain how MSNs enhance

biosensor performance.

1. Multimodal Detection: Discuss the possibilities of multimodal detection using

MSNs.

vii. Label-Free Detection: Highlight the potential for label-free detection methods.

viii. Standardization and Interoperability: Address the need for standardization and

interoperability in biosensor development.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

This manuscript by Álvaro de Jesús Ruíz-Baltazar et al., focuses on the Ag- decorated Fe3O4 nanoparticles. It provides a comprehensive and well-structured evaluation of the cytotoxicity and potential applications of the nanoparticles, especially in HFF-1 and MCF-7. The research utilize a combination of SEM, XRD and XPS to eliminate the physics and chemical properties of the nanoparticles, and give the details of how to synthesis those nanoparticles. Through magnetic assays, the nanoparticles exhibit superparamagnetic behavior which is good for the biomedical applications. They also use experiments to demonstrate its high biocompatibility with human fibroblasts and breast cancer cells, and highlight their promising therapeutic efficacy through detailed analysis and robust experimental methodologies. Overall, this is meaningful research that could shed light on the cancer treatment by nanoparticles. The data is solid, and the structure is easy to follow. Key message is presented clearly. I would recommend this manuscript to be published after the author address some minor points listed below:

1.      Figures are not professionally made. A new version with high resolution for all figs are needed.

2.      SEM results in Fig. 1 is not clear. Hard to identify the structure of Ag-decorated Fe3O4. Maybe a illustration figure could help the readers to understand.

3.      What is the sharp peak at 0 in Fig. 2(f)? Also remember that this 2(f) need a axis label.

4.      Could the authors explore the potential of combining Fe3O4-Ag nanoparticles with other therapeutic agents to enhance their efficacy?

5.      What are the potential limitations of the current study? Will this nanoparticle’s efficiency decrease with aging? What measures were taken to ensure the uniformity and stability of the synthesized nanoparticles over time?

 

6.      What are the potential in vivo implications of the reduced ROS production observed with Fe3O4-Ag nanoparticles?

Comments on the Quality of English Language

This manuscript overall is well structured and presented clearly.

Author Response

RESPONSE TO REVIEWER 3

 

Comments and Suggestions for Authors

This manuscript by Álvaro de Jesús Ruíz-Baltazar et al., focuses on the Ag- decorated Fe3O4 nanoparticles. It provides a comprehensive and well-structured evaluation of the cytotoxicity and potential applications of the nanoparticles, especially in HFF-1 and MCF-7. The research utilize a combination of SEM, XRD and XPS to eliminate the physics and chemical properties of the nanoparticles, and give the details of how to synthesis those nanoparticles. Through magnetic assays, the nanoparticles exhibit superparamagnetic behavior which is good for the biomedical applications. They also use experiments to demonstrate its high biocompatibility with human fibroblasts and breast cancer cells, and highlight their promising therapeutic efficacy through detailed analysis and robust experimental methodologies. Overall, this is meaningful research that could shed light on the cancer treatment by nanoparticles. The data is solid, and the structure is easy to follow. Key message is presented clearly. I would recommend this manuscript to be published after the author address some minor points listed below:

1. Figures are not professionally made. A new version with high resolution for all figs are needed.

RESPONSE:

The authors appreciate your comments and have addressed your observations with great emphasis. Accordingly, all figures have been improved as much as possible. In the case of the graphs, they have been re-plotted using software that enhances the resolution and quality of these images.

2. SEM results in Fig. 1 is not clear. Hard to identify the structure of Ag-decorated Fe3O4. Maybe a illustration figure could help the readers to understand.

RESPONSE:

A scheme of the distribution of the Fe3O4 and Ag nanostructures is shown in Figure 1 (e), which is included in this new version of the manuscript.

3. What is the sharp peak at 0 in Fig. 2(f)? Also remember that this 2(f) need a axis label.

RESPONSE:

In this case, the intensity peak at 0.22 is associated with the carbon present in the grid used to mount the sample for microscopy analysis. Therefore, it was decided to exclude it from the analysis to avoid overshadowing and to highlight the presence of the elements of interest, which are Fe, O, and Ag. Additionally, the intensity observed at 1.5 eV is associated with the aluminum of the sample holder. As an important note, the sample holder is an aluminum cylinder, and a carbon tape was placed on it to mount the Fe3O4-Ag sample. (Gold or copper-coated grids were not used to ensure that the signals of the Fe, O, and Ag elements are clearly shown).

 

The axis of the EDX analysis graph has already been included, and the units are in eV.

4. Could the authors explore the potential of combining Fe3O4-Ag nanoparticles with other therapeutic agents to enhance their efficacy?

We appreciate this comment from the reviewer. Indeed, one of our short-term objectives is to explore future antiproliferative applications in vitro and in vivo using different cancer models. However, ensuring their safety is paramount before evaluating the cytotoxic effects of nanoparticles in combination with other drugs or bioactive molecules. Therefore, one of our initial approaches has been to conduct nanotoxicological evaluations of these systems. To achieve this, we employed four standard bioassays to determine the safety of these nanomaterials at their respective concentrations. If safety concerns arise, our goal is to propose new concentration ranges to ensure their safe utilization.

 

5. What are the potential limitations of the current study? Will this nanoparticle’s efficiency decrease with aging? What measures were taken to ensure the uniformity and stability of the synthesized nanoparticles over time?

What are the potential limitations of the current study?

RESPONSE:

• The possible limitation of the current study could be that the synthesis of Fe3O4-Ag. Structures with a high degree of uniformity in terms of morphology and particle size distribution presents certain challenges. Although the nanoparticles reported in this manuscript are very stable and acceptably uniform, optimization in the synthesis of such structures is an ongoing challenge and can always be improved. It is important to note that some synthesis methods generate uniform structures; However, the chemical reagents used can sometimes decrease the reactivity of nanoparticle surfaces because the surfactants are difficult to remove. In contrast, the synthesis method presented in this manuscript is more environmentally friendly and does not involve the use of surfactants or complex colloidal media that may interfere with the surface integrity of the nanoparticles. Therefore, the limitation, in general for this nanostructures type could be the synthesis of these materials.

Will this nanoparticle’s efficiency decrease with aging?

RESPONSE

• Currently we have not observed a decrease in particle efficiency as a function of aging. In previous studies, these same nanoparticles have been used and over time no decrease in their efficiency has been observed. However, to answer this question in depth, similar studies would need to be performed after a long or relatively long period of “aging” of the nanostructures. These studies could be considered in subsequent works. The authors appreciate your suggestions.

 

What measures were taken to ensure the uniformity and stability of the synthesized nanoparticles over time?

RESPONSE

 

• To ensure the uniformity of the nanoparticles, special care has been taken on the reproducibility of the experiments. This has allowed the synthesized particles to be uniform in terms of morphology, size and composition. The working group has extensive experience with this type of nanoparticles and has been able to control the uniformity of the particles as much as possible. Regarding the stability of the synthesized nanoparticles over time, our research group has observed that the synthesized particles have retained their properties for several months and even a couple of years. One measure to maintain its stability over the years is to preserve the particles in powder form, since this prevents them from reacting with the colloidal medium if they are kept in solution.

 

6. What are the potential in vivo implications of the reduced ROS production observed with Fe3O4-Ag nanoparticles?

 

RESPONSE

We appreciate the reviewer's insightful comment. One of the primary mechanisms of metal nanoparticles' and metal oxides' toxicity is their ability to induce overproduction of ROS, thereby initiating cellular damage processes and, ultimately, apoptosis. The observed reduction in ROS at a single concentration in this study might be compensated for in an in vivo system. Furthermore, it is worth noting that these magnetite nanosystems are designed to act in combination with various drugs and bioactive molecules possessing antiproliferative effects. Therefore, the slight reduction in ROS levels observed in cells before conjugation with antineoplastic drugs suggests their potential biosafety at a systemic level. However, this can only be definitively evaluated through in vivo assays.

To point out this, we added the following idea in the manuscript:

“The production of ROS was measured, as shown in Figure 7, to corroborate the biocompatibility of Fe₃O₄ and Fe₃O₄-Ag decorated nanoparticles in human cells. Different concentrations of nanoparticles were tested in human fibroblast cells HFF-1 (Figure 7a). Interestingly, only higher concentrations (100 µg/mL) of Fe3O4-Ag decorated nanoparticles reduced ROS production. This finding contrasts with established knowledge, which typically associates magnetite nanoparticles with inducing oxidative stress in mammalian cells(Horie & Tabei, 2021; Paunovic et al., 2020). The same effect was observed in human breast cancer MCF-7 cells, as illustrated in Figure 7b, Fe₃O₄-Ag decorated nanoparticles induced a decrease in the level of ROS production only after 50 µg/mL, and at higher concentrations (100 µg/mL) Fe₃O₄ also reduced the level of ROS production. “To the best of our knowledge, Fe3O4 nanoparticles are widely recognized for inducing oxidative stress effects in cells. However, a report indicates that Fe3O4 magnetite nanoparticles demonstrate the opposite effect: they reduce metal-induced oxidative stress by decreasing malondialdehyde concentration and increasing superoxide dismutase levels(Konate et al., n.d.)”

 

 

 

 

 

Comments on the Quality of English Language

This manuscript overall is well structured and presented clearly.

Author Response File: Author Response.pdf