Cytotoxicity studies of Fe3O4 nanoparticles in chicken macrophage cells

Magnetic Fe3O4 nanoparticles (Fe3O4-NPs) have been widely investigated for their biomedical applications. The main purpose of this study was to evaluate the cytotoxic effects of different sizes of Fe3O4-NPs in chicken macrophage cells (HD11). Experimental groups based on three sizes of Fe3O4-NPs (60, 120 and 250 nm) were created, and the Fe3O4-NPs were added to the cells at different doses according to the experimental group. The cell activity, oxidative index (malondialdehyde (MDA), superoxide dismutase (SOD) and reactive oxygen species (ROS)), apoptosis and pro-inflammatory cytokine secretion level were detected to analyse the cytotoxic effects of Fe3O4-NPs of different sizes in HD11 cells. The results revealed that the cell viability of the 60 nm Fe3O4-NPs group was lower than those of the 120 and 250 nm groups when the same concentration of Fe3O4-NPs was added. No significant difference in MDA was observed among the three Fe3O4-NP groups. The SOD level and ROS production of the 60 nm group were significantly greater than those of the 120 and 250 nm groups. Furthermore, the highest levels of apoptosis and pro-inflammatory cytokine secretion were caused by the 60 nm Fe3O4-NPs. In conclusion, the smaller Fe3O4-NPs produced stronger cytotoxicity in chicken macrophage cells, and the cytotoxic effects may be related to the oxidative stress and apoptosis induced by increased ROS production as well as the increased expression of pro-inflammatory cytokines.

xii) The text within Figure 8 is not visible. It should be presented in a proper way. Apoptosis cell % is not visible in the diagram and histogram. xiii) Apoptosis data does not corroborate with the cell viability data. Cell viability shows almost biocompatible nature of 120 nm NPs except at 50 µg/mL dose. But apoptosis study exhibited higher % apoptosis for 120 nm NPs treatment at all concentrations as compared to control untreated cells.
Why is there reduction in % apoptosis with increasing concentration of 120 nm NPs?
xiv) The English should be more polished.
xv) Conclusion section is poorly written.

Review form: Reviewer 2
Is the manuscript scientifically sound in its present form? Yes

Recommendation?
Major revision is needed (please make suggestions in comments)

Comments to the Author(s)
The manuscripts reveals an interesting study of cytotoxicity of Fe3)4 nano particles on chicken macrophage cells to assess its effectiveness again S. Entreditis infection in poultry. The report reveals that cytotoxicity of 250 nm particles is least and the particle of this size may be used as an antibiotic alternative. However, I have some concerns if such big size nanoparticles can be used in drug delivery and authors must add some literature reports wherein particles greater than 200 nm has been used. Authors should also convince readers about the effectiveness of the 250 nm Fe3O4 nanoparticles to cross blood brain barrier since mostly for the drug delivery nanoparticle size used is between 10-100 nm.

Review form: Reviewer 3
Is the manuscript scientifically sound in its present form? Yes The authors describe research that utilized magnetic nanoparticles of 60, 120 and 250 nm diameters. They provide a general approach for how the nanoparticles were prepared but not how the approach was used to get just 60 or 120 or 250 nm particles. This point is fairly important in the event that someone wants to reproduce the authors work.
The authors note that toxicity of nanoparticles should be examined prior to their use in vivo (e.g., p 7, lines 29-31, 'Therefore, it is important to fully investigate the biosafety of nanoparticles before they are used in biomedical or livestock production.' What is the fate of the nanoparticles? This is relevant here because if they are retained and the livestock eaten, they may also affect humans and so toxic effects in humans also needs to be examined. The expected use of these iron nanoparticles should be stated. The authors conclude the manuscript with the statement 'Based on this study, 250 nm Fe3O4-NPs have good biological safety and maybe be used in disease diagnosis and substitution research in poultry industry.' (p. 9, conclusion section, lines 39-41). Are the authors suggesting the nanoparticles are going to be used to treat infections and take advantage of the ROS generation or, will the particles simply act as carriers. In the latter case, the particles studied in this manuscript would not be relevant as there surfaces were unmodified. The authors, at a minimum, need to describe how the nanoparticles are expected to be used. They may also want to state that in vivo degradation of particles with coatings are bearing 'payloads' (e.g., drugs) may result in the bare magnetic nanoparticles.

Misc:
Abstract: The sentence 'The results revealed that the cell viability of the 250 nm Fe3O4-NPs group was significantly greater (P < 0.05) than that of the 60 nm and 120 nm groups at a concentration of 50 μg/mL, and the activity level of the 120 nm and 250 nm groups was significantly greater (P < 0.01) than that of the 60 nm group at a concentration of 100 μg/mL.' is poorly written/confusing. If I understand it correctly, the authors are simply providing a rank order and could say that the smaller the particle size the greater the activity. If they feel it necessary, the qualifiers regarding concentration, etc. can be added. The editor assigned to your manuscript has now received comments from reviewers. We would like you to revise your paper in accordance with the referee and Subject Editor suggestions which can be found below (not including confidential reports to the Editor). Please note this decision does not guarantee eventual acceptance.
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I have some serious concerns and doubt on the results and interpretation especially in the biology part. Besides scientific and technical issues, it seems that the manuscript appears like a draft and it is not suitable for publication in "Royal Society Open Science". Therefore, it should be rejected. Some specific concerns: i) Explanation of all the data is poor.
ii) '…there are still some controversial results concerning the cytotoxic effects from use of SPIONs…' The controversial results should be described in Introduction section.
iii) The synthesis procedure did not clarify the conditions of formation of three different sized (60 nm, 120 nm, 250 nm) nanoparticles. iv) At which wavelength the absorbance of MDA/SOD was measured using ELISA? It should be mentioned in experimental section. v) Fluorescence microscopy or confocal microscopy would be more convincing for determination of ROS. The present work lacks of state-of-the art techniques. vi) Why is there no much difference in size of NPs obtained from TEM and DLS, especially for 120 nm and 250 nm NPs? Generally, the hydrodynamic size of NPs obtained in DLS is much greater than the size of NPs obtained in TEM.
vii) The interpretation of cell viability data ( Figure 4) and the execution of the experiment are faulty. Although 60 nm NPs exerted slight cytotoxicity in HD11 cells at 50-100 µg/mL as compared to control untreated cells, it did not exert any cytotoxicity (almost nil) even at higher dose 200 µg/mL. Again at 400 µg/mL dose, it exhibited similar slight toxicity as observed for 50 µg/mL dose. So the result is not consistent as per the dosing.
It seems that all the NPs (60 nm, 120 nm and 250 nm) are showing very slight toxicity, as compared to untreated control cells at a broad concentration range (50-400 µg/mL). However, there is no dose dependent consistent result for 60 nm NPs, from which it can be interpreted that 60 nm NPs are more toxic than others. e.g. at 200 µg/mL dose, 250 nm NPs are more toxic than 60 nm NPs and at 400 µg/mL dose, the toxicity for 60 nm and 250 nm NPs are similar. Therefore, the interpretations of further study results which are based on the cell viability data are also misleading.
viii) The cytotoxic potential of 60 nm NPs at 200 µg/mL is lower than that of 50 and 100 µg/mL dose. Why did it happen? ix) Why did you check MDA, SOD and ROS levels and what is the outcome of these data with respect to the cytotoxicity potential of NPs? It should be clearly explained in corresponding result sections.
x) How did the self-regulation of cells influence the higher SOD level for the treatment with 60 nm NPs, unlike other NPs? xi) Why is the ROS level gradually reduced with increasing concentration of 60 nm NPs? The result is absurd.
xii) The text within Figure 8 is not visible. It should be presented in a proper way. Apoptosis cell % is not visible in the diagram and histogram. xiii) Apoptosis data does not corroborate with the cell viability data. Cell viability shows almost biocompatible nature of 120 nm NPs except at 50 µg/mL dose. But apoptosis study exhibited higher % apoptosis for 120 nm NPs treatment at all concentrations as compared to control untreated cells.
Why is there reduction in % apoptosis with increasing concentration of 120 nm NPs?
xiv) The English should be more polished.
xv) Conclusion section is poorly written.

Reviewer: 2
Comments to the Author(s) The manuscripts reveals an interesting study of cytotoxicity of Fe3)4 nano particles on chicken macrophage cells to assess its effectiveness again S. Entreditis infection in poultry. The report reveals that cytotoxicity of 250 nm particles is least and the particle of this size may be used as an antibiotic alternative. However, I have some concerns if such big size nanoparticles can be used in drug delivery and authors must add some literature reports wherein particles greater than 200 nm has been used. Authors should also convince readers about the effectiveness of the 250 nm Fe3O4 nanoparticles to cross blood brain barrier since mostly for the drug delivery nanoparticle size used is between 10-100 nm.
Reviewer: 3 Comments to the Author(s) Manuscript ID: RSOS-191561 Title: Cytotoxicity studies of Fe3O4 nanoparticles in chicken macrophage cells Authors: Zhang, S., et al.
This manuscript describes the preparation of Magnetic Fe3O4 nanoparticles and examines them for their activity (oxidative index (malondialdehyde, superoxide dismutase and reactive oxygen species formation), apoptosis and pro-inflammatory cytokine secretion level in chicken macrophage cells as a function of nanoparticle size. They find that, when there are differences, there is greater activity in smaller nanoparticles.
The authors describe research that utilized magnetic nanoparticles of 60, 120 and 250 nm diameters. They provide a general approach for how the nanoparticles were prepared but not how the approach was used to get just 60 or 120 or 250 nm particles. This point is fairly important in the event that someone wants to reproduce the authors work.
The authors note that toxicity of nanoparticles should be examined prior to their use in vivo (e.g., p 7, lines 29-31, 'Therefore, it is important to fully investigate the biosafety of nanoparticles before they are used in biomedical or livestock production.' What is the fate of the nanoparticles? This is relevant here because if they are retained and the livestock eaten, they may also affect humans and so toxic effects in humans also needs to be examined. The expected use of these iron nanoparticles should be stated. The authors conclude the manuscript with the statement 'Based on this study, 250 nm Fe3O4-NPs have good biological safety and maybe be used in disease diagnosis and substitution research in poultry industry.' (p. 9, conclusion section, lines 39-41). Are the authors suggesting the nanoparticles are going to be used to treat infections and take advantage of the ROS generation or, will the particles simply act as carriers. In the latter case, the particles studied in this manuscript would not be relevant as there surfaces were unmodified. The authors, at a minimum, need to describe how the nanoparticles are expected to be used. They may also want to state that in vivo degradation of particles with coatings are bearing 'payloads' (e.g., drugs) may result in the bare magnetic nanoparticles.

Misc:
Abstract: The sentence 'The results revealed that the cell viability of the 250 nm Fe3O4-NPs group was significantly greater (P < 0.05) than that of the 60 nm and 120 nm groups at a concentration of 50 μg/mL, and the activity level of the 120 nm and 250 nm groups was significantly greater (P < 0.01) than that of the 60 nm group at a concentration of 100 μg/mL.' is poorly written/confusing. If I understand it correctly, the authors are simply providing a rank order and could say that the smaller the particle size the greater the activity. If they feel it necessary, the qualifiers regarding concentration, etc. can be added.

Do you have any ethical concerns with this paper? No
Have you any concerns about statistical analyses in this paper? No

Recommendation?
Accept as is Thank you very much for your hard work and valuable comments on our paper (Manuscript ID: RSOS-191561). We feel lucky that our manuscript went to these reviewers, as their comments not only helped us with the improvement of our manuscript but also offered some unique ideas for further studies. We have studied the comments carefully and have made corrections that we hope will be met with approval.
Please find below details regarding all of the changes we have made to the manuscript in response to the editor's and reviewers' comments. All of the changes are marked in red in both this response letter and the revised manuscript.
Reply to Reviewer 1: i) Explanation of all the data is poor.
Reply: Thank you for your insightful suggestion. We have reinterpreted all of the data in parts 3.2, Figure 4, the cell viability of the 250 nm Fe3O4-NPs group was significantly greater than those of the 60 nm and 120 nm groups at the concentration of 50 μg/mL, and the activity levels of the 120 nm and 250 nm groups were significantly greater than that of the 60 nm group at a concentration of 100 μg/mL."; Part 3.3, "SOD levels of the 60 nm group were significantly higher than those of the 120 nm and 250 nm groups at 50 μg/mL. SOD levels of the 60 nm group were significantly higher, showing extreme differences, than those of the 120 nm and 250 nm groups at 100, 200 and 400 μg/mL."; Part 3.4, "At concentrations of 50 and 100 μg/mL, the Appendix A extent of apoptosis of the 250 nm group was significantly less than those of the 60 nm and 120 nm groups. At a concentration of 200 μg/mL, the extent of apoptosis of the 120 nm group was significantly less than that of the 60 nm group, and the extent of apoptosis of the 250 nm group was extremely significantly less than that of the 60 nm group. Additionally, the extent of apoptosis of the 250 nm group was significantly lower than that of the 60 nm group at 400 μg/mL.") ii) '…there are still some controversial results concerning the cytotoxic effects from use of

SPIONs…'
The controversial results should be described in Introduction section.  Med. Chem. 15, 1914-1929. (doi: 10.2174 iii) The synthesis procedure did not clarify the conditions of formation of three different sized (60 nm, 120 nm, 250 nm) nanoparticles.
Reply: Thank you for your insightful suggestion. Revisions have been made according to this suggestion in part 2.1. (Part 2.1, "Fe3O4-NPs with diameters of approximately 60 nm, 120 nm and 250 nm were prepared by a hydrothermal method with FeCl3 and NaAc•3H2O as raw materials. A 0.4 g or 0.6 g sample of FeCl3 and 3.6 g of NaAc•3H2O were added into the mixed solvent of 10 mL of glycol and 30 mL of diethylene glycol and stirred until fully dissolved by ultrasound, and these solutions were used to prepare 60 nm or 120 nm Fe3O4-NPs. A 0.82 g sample of FeCl3 and 3.6 g of NaAc•3H2O were added into 40 mL of glycol and stirred to achieve full dissolution by ultrasound, which was used to prepare the 250 nm Fe3O4-NPs. After complete dissolution, the mixture was transferred into a 50 mL Teflon-sealed autoclave and heated at 200 °C for 12 h.") iv) At which wavelength the absorbance of MDA/SOD was measured using ELISA? It should be mentioned in experimental section.
Reply: Thank you for your insightful suggestion. Revisions have been made according to this suggestion in part 2.4. (Part 2.4, "The absorbance of MDA/SOD was measured at 532 nm/450 nm with a microplate reader (Infinite M200 Pro, Tecan, Switzerland).") v) Fluorescence microscopy or confocal microscopy would be more convincing for determination of ROS. The present work lacks of state-of-the art techniques.
Reply: Thank you for your insightful suggestion. In view of the actual situation and related literature [1][2][3], it is also feasible to use the reactive oxygen species assay kit to detect ROS. 1. Chen S, Chen S, Zeng Y, Lin L, Wu C, Ke Y, Liu G. 2018 Size-dependent superparamagnetic iron oxide nanoparticles dictate interleukin-1beta release from mouse bone marrow-derived macrophages. J. Appl. toxicol. 38, 978-986. (doi: 10.1002/jat.3606) vii) The interpretation of cell viability data ( Figure 4) and the execution of the experiment are faulty.
Although 60 nm NPs exerted slight cytotoxicity in HD11 cells at 50-100 µg/mL as compared to control untreated cells, it did not exert any cytotoxicity (almost nil) even at higher dose 200 µg/mL. Again at 400 µg/mL dose, it exhibited similar slight toxicity as observed for 50 µg/mL dose. So the result is not consistent as per the dosing.
It seems that all the NPs (60 nm, 120 nm and 250 nm) are showing very slight toxicity, as compared to untreated control cells at a broad concentration range (50-400 µg/mL). However, there is no dose dependent consistent result for 60 nm NPs, from which it can be interpreted that 60 nm NPs are more toxic than others. e.g. at 200 µg/mL dose, 250 nm NPs are more toxic than 60 nm NPs and at 400 µg/mL dose, the toxicity for 60 nm and 250 nm NPs are similar. Therefore, the interpretations of further study results which are based on the cell viability data are also misleading.
Reply: Thank you for your insightful suggestion. The main purpose of this study was to evaluate the cytotoxic effects of different sizes of Fe3O4-NPs in HD11 cells, and we analysed the cytotoxic effects of different sizes of Fe3O4-NPs on cell activity at the same concentration with the analysis of LDS's one-way analysis of variance (ANOVA). Since I did not describe this clearly in the previous paper, the data description has now been corrected in parts 3.2, 3.3 and 3.4 (same as the answer to the first question).
viii) The cytotoxic potential of 60 nm NPs at 200 µg/mL is lower than that of 50 and 100 µg/mL dose. Why did it happen?
Reply: Thank you for your insightful suggestion. We analysed the cytotoxic effects of different sizes of Fe3O4-NPs on the cell activity at the same concentration, and the cytotoxic effects of different concentrations (50, 100, 200 and 400 µg/mL) of 60 nm Fe3O4-NPs on cell activity were not significant (P > 0.05). Additionally, since I did not describe this clearly in the previous paper, the data description has now been corrected in part 3.2 (same as the answer to the first question). Reply: Thank you for your insightful suggestion. In this study, it can be seen that "the ROS level gradually decreased with increasing concentrations of 60 nm Fe3O4-NPs". At the same time, we should also notice that "the SOD level gradually increased with increasing concentrations of 60 nm Fe3O4-NPs". SOD is a scavenger of oxygen free radicals. With increasing concentrations of 60 nm NPs, the self-regulation of cells may stimulate SOD activity in response to increased oxidative stress by ROS.
xii) The text within Figure 8 is not visible. It should be presented in a proper way. Apoptosis cell % is not visible in the diagram and histogram.
Reply: Thank you for your insightful suggestion. I will upload Figure 8 again in a more appropriate manner. The percentage of apoptotic cells is visible in the histogram. xiii) Apoptosis data does not corroborate with the cell viability data. Cell viability shows almost biocompatible nature of 120 nm NPs except at 50 µg/mL dose. But apoptosis study exhibited higher % apoptosis for 120 nm NPs treatment at all concentrations as compared to control untreated cells.
Why is there reduction in % apoptosis with increasing concentration of 120 nm NPs?
Reply: Thank you for your insightful suggestion. We analysed the cytotoxic effects of different sizes of Fe3O4-NPs on cell activity and apoptosis at the same concentration. Additionally, because I did not describe this clearly in the previous version, the data description has now been corrected in parts 3.2, 3.3 and 3.4 (same as the answer to the first question) xiv) The English should be more polished.
Reply: Thank you for your insightful suggestion. The editors at AJE have polished our manuscript.
And the editing certificate is as follows.
xv) Conclusion section is poorly written.
Reply: The cell viability, oxidation index (MDA, SOD and ROS) and apoptosis level were detected to analyse the cytotoxicity of Fe3O4-NPs of different sizes in HD11 cells. By referring to the articles about the cytotoxicity of Fe3O4-NPs, we selected these indicators for detection and repeated verification in chicken HD11 cells, and thus, we obtained the conclusion in this paper. Through reinterpretation of all the data in part 3, the conclusion of this study was obvious. The conclusion was that the smaller Fe3O4-NPs produced stronger cytotoxicity in chicken macrophage cells, which may be related to the oxidative stress and apoptosis induced by increased ROS production as well as the increased expression of pro-inflammatory cytokines. The 250 nm Fe3O4-NPs have good biological safety and may be used in disease diagnosis and substitution research in the poultry industry.
Reply to Reviewer 2: The report reveals that cytotoxicity of 250 nm particles is least and the particle of this size may be used as an antibiotic alternative. However, I have some concerns if such big size nanoparticles can be used in drug delivery and authors must add some literature reports wherein particles greater than 200 nm has been used. Authors should also convince readers about the effectiveness of the 250 nm Fe3O4 nanoparticles to cross blood brain barrier since mostly for the drug delivery nanoparticle size used is between 10-100 nm.
Reply: Thank you for your insightful suggestion. In this study, 250 nm Fe3O4-NPs have good biological safety, which lays a foundation for the potential application of Fe3O4-NPs in the poultry industry. However, Fe3O4-NPs as potential antibiotic substitutes and their mechanism require further experiments. Shi et al. reported that the 200 nm Fe3O4-NPs may be a potential antibiotic alternative to control Salmonella enteritidis (S. enteritidis) infection during clinical therapy and in poultry industry operations [1]. Our previous research found that Fe3O4-NPs could be observed in the livers of chickens, indicating that they could pass through the blood brain barrier [2]. The authors describe research that utilized magnetic nanoparticles of 60, 120 and 250 nm diameters.
They provide a general approach for how the nanoparticles were prepared but not how the approach was used to get just 60 or 120 or 250 nm particles. This point is fairly important in the event that someone wants to reproduce the authors work.
Reply: Thank you for your insightful suggestion. Revisions have been made according to this suggestion in part 2.1. (Part 2.1, "Fe3O4-NPs with diameters of approximately 60 nm, 120 nm and 250 nm were prepared by a hydrothermal method with FeCl3 and NaAc•3H2O as raw materials. A 0.4 g or 0.6 g sample of FeCl3 and 3.6 g of NaAc•3H2O were added into the mixed solvent of 10 mL of glycol and 30 mL of diethylene glycol and stirred until fully dissolved by ultrasound, and these solutions were used to prepare 60 nm or 120 nm Fe3O4-NPs. A 0.82 g sample of FeCl3 and 3.6 g of NaAc•3H2O were added into 40 mL of glycol and stirred to achieve full dissolution by ultrasound, which was used to prepare the 250 nm Fe3O4-NPs. After complete dissolution, the mixture was transferred into a 50 mL Teflon-sealed autoclave and heated at 200 °C for 12 h.") The authors note that toxicity of nanoparticles should be examined prior to their use in vivo (e.g., p 7, lines 29-31, 'Therefore, it is important to fully investigate the biosafety of nanoparticles before they are used in biomedical or livestock production.' What is the fate of the nanoparticles? This is relevant here because if they are retained and the livestock eaten, they may also affect humans and so toxic effects in humans also needs to be examined. Reply: Thank you for your insightful suggestion. There are some reports on the cytotoxicity of Fe3O4 nanoparticles in mammals (rats, mice) or humans [1][2][3][4]. Poultry has many different characteristics in structure and function from mammals, and HD11 cells share many similarities with normal chicken macrophages and have the advantages of rapid subculture growth; hence, they can be used as an ideal model of chicken macrophages for in vitro studies [5][6][7]. In this paper, we mainly evaluated the cytotoxicity of Fe3O4-NPs in HD11 cells at the cellular level in order to select the relatively safe size of Fe3O4-NPs. In addition, we will carry out animal experiments to verify the safety and residual problems of Fe3O4-NPs. Thank you for your understanding.
1. Lee JH, Ju JE, Kim BI, Pak PJ, Choi EK, Lee HS, Chung N. 2014 Rod-shaped iron oxide nanoparticles are more toxic than sphere-shaped nanoparticles to murine macrophage cells.