Understanding Macrophage Interaction with Antimony-Doped Tin Oxide Plasmonic Nanoparticles

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript reports of very preliminary experiments on macrophage response towards exposure to nanoparticles amenable for phothermal treatment.

The authors have focused on excluding contamination by endotoxins - there couls be other types of contamination, also by metals or other agents. A discussion of this should be beneficial.

The authors showed how the macrophages changed some of their features upon exposure and activation of the nanopparticles, but they have not showed or indicated how this is coherent with a possible therapeutic role of the macrophages against cancer cells.

There is no discussion of the possible stability of NPs within cells and release of metal ions, with the predcitable consequences.

Reading the conclusions, it has not been shown sufficiently that the assays described in the papers are giving the information required for programming future experiments. The relevance of these preliminary tests (desirable macrophage responses) should be discussed with more caution.

 

Some sentences in the text are quite difficult to follow, thinking about an audience of non experts - e.g. L37-40, L220-251. Maybe a re-reading of the whole paper could be beneficial, thinking to a potential audience interested in the biological effects.

Also the abstract could be made easier to understand.

Figure legends are always missing details which could make them understandable without reading the whole paper - too many abbreviations and no explanations.

 

 

 

L89 I think that sentences should never start with abbreviations, symbols or numbers

L94 I think it is esential to express centrifugation speed in xg, unless a full description of the centrifuge dimensions and features is given

L102, L109, L121 - for equipments and kits it is better to report in parentheses the model, brand name, city and country

L128-129, L134 please explain these concentrations - v/v from a stock solution? there is no way of understanding the quantities

L138 I think some abbreviations have not been defined

L144 pay attention to measure units - mL

L178 complete the indication on concentration

L183-185 details are missing here

L235 I do not see the size distribution in the figure

L273-274 is there any way to show that indeed the thermal gradient is getting larger, with numerical indications?

L281-282 and Table S3 - I am not familiar with this assay but surely there cannot be so many significant digits in these measurements? The text already reports the values, so why include the Table?

L327 there are  no "cells" in the red boxes

L335 how can the authors demonstrate that these are indeed the ATO NPs? some identification should be possible with EDX analysis as in Figure S2? Figure 4B is not clear about what can be seen and identified as NPs

L352-357 highly speculative; no changes were evidenced in Figure 2A

L384 a part of Figure S3B is not visible in the supplementary file

L411 "professional phagocytes" cannot be accepted

L422-424 I do no think this has been shown in the paper

L426-428 rephrase - the metabolic activity is shown by cells, not by NPs

 

Comments on the Quality of English Language

The English language is very good - nonetheless some parts of the paper are very difficult to read thinking to a potential audience interested in "cells"

Author Response

 

1. The manuscript reports of very preliminary experiments on macrophage response towards exposure to nanoparticles amenable for phothermal treatment.

We appreciate the reviewer's concern regarding the preliminary nature of our experiments. Our study was designed to establish a foundational understanding of the interactions between ATO nanoparticles and macrophages, which is critical before advancing to more complex in vivo models or therapeutic applications.

The study was designed as a proof-of-concept to demonstrate the feasibility of macrophage uptake of ATO nanoparticles and to explore their photothermal properties within these cells. The results serve as a crucial first step in establishing the potential of ATO nanoparticles for photothermal therapy, particularly in the context of their interaction with immune cells.

The choice of J774A.1 macrophages was based on their well-established role as a model system for studying nanoparticle-cell interactions. Their phagocytic activity and consistent response to stimuli make them a suitable model for investigating cellular uptake and response to ATO nanoparticles. Nanoparticle concentrations were selected based on a preliminary dose-response study to identify levels that induced observable effects without causing significant cytotoxicity. These concentrations align with those reported in previous studies using similar nanoparticles, which have been cited in the manuscript.

2. The authors have focused on excluding contamination by endotoxins - there couls be other types of contamination, also by metals or other agents. A discussion of this should be beneficial.

We appreciate the reviewer's comment regarding potential contaminants beyond endotoxins. While this study focused on endotoxin contamination due to its known impact on immune responses, we acknowledge the importance of considering other potential contaminants.  It is important to note that ATO NPs are known for their exceptional thermal and corrosive stability, as demonstrated in their application in solar concentrators and catalysts. No ion release has been observed under these extreme conditions or in our study.  Both metallic species are simultaneously detected or missed in the cell’s areas probed. See the revised FIB-SEM-EDX section:

EDX analysis on a cell cross-section at four distinct FIB-SEM locations revealed the chemical fingerprint of the ATO NPs (25 µg/mL) following uptake, as shown in Fig S2. EDX spectra (Fig S2 (points 3-4)) show the tin (Sn) and antimony (Sb) signals, which confirm the presence of ATO NPs. That is also evidence of the stability of ATO NPs, as EDX signals from antimony and tin are always coupled. In the case of a metal ion solvation, one would expect the signals decoupling and, if both metals are dissolved, homogenous distribution of Sn and Sb throughout the entire examined cell, which was not observed. That was predictable as selected ATO materials can withstand high temperatures in solar concentrators [27] and corrosive media [28].

 

3. The authors showed how the macrophages changed some of their features upon exposure and activation of the nanoparticles, but they have not showed or indicated how this is coherent with a possible therapeutic role of the macrophages against cancer cells.

Thank you for your valuable feedback. We acknowledge that our current study primarily focuses on preliminary experiments concerning the macrophage response to ATO NPs. Our research intended to lay a foundational understanding of how these NPs interact with macrophages, which is a critical step before advancing to more comprehensive in vivo studies.

In response to your concern about the therapeutic role of macrophages, we have added a discussion highlighting the potential implications of our findings. Specifically, we noted that the observed macrophage changes, such as increased uptake and possible shifts in activation states, could be leveraged to enhance the therapeutic efficacy of macrophages in targeting cancer cells. Further research is needed to fully elucidate how these macrophage interactions may translate into improved anti-cancer effects, including in vivo studies to explore these possibilities in a more complex biological context.

We appreciate your suggestion to evaluate the potential therapeutic applications of ATO NPs more thoroughly and will consider it as we progress to future research phases.

Here is the revised discussion that was included in the manuscript.

Importantly, the observed changes in macrophage behavior—such as increased uptake of ATO NPs and potential shifts in activation states—suggest that these nanoparticles might be leveraged to enhance the therapeutic efficacy of macrophages in targeting cancer cells. For example, ATO NPs could induce macrophages to adopt a more pro-inflammatory M1 phenotype, known to have anti-tumor effects. This shift could potentially improve the effectiveness of photothermal therapy by targeting cancer cells directly and enhancing the overall immune response within the tumor microenvironment. Further research is needed to explore how these macrophage changes translate into improved anti-cancer effects, including in vivo studies that assess the inter-action between ATO NPs, macrophages, and tumor cells.

4. There is no discussion of the possible stability of NPs within cells and release of metal ions, with the predcitable consequences.

We appreciate the reviewer's comment regarding stability of NPs within cells and release of metal ions. This is addressed in one of the previous comments in FIB-SEM-EDX discussion:

EDX analysis on a cell cross-section at four distinct FIB-SEM locations revealed the chemical fingerprint of the ATO NPs (25 µg/mL) following uptake, as shown in Fig S2. EDX spectra (Fig S2 (points 3-4)) show the tin (Sn) and antimony (Sb) signals, which confirm the presence of ATO NPs. That is also evidence of the stability of ATO NPs, as EDX signals from antimony and tin are always coupled. In the case of a metal ion solvation, one would expect the signals decoupling and, if both metals are dissolved, homogenous distribution of Sn and Sb throughout the entire examined cell, which was not observed. That was predictable as selected ATO materials can withstand high temperatures in solar concentrators [27] and corrosive media [28].”

5. Reading the conclusions, it has not been shown sufficiently that the assays described in the papers are giving the information required for programming future experiments. The relevance of these preliminary tests (desirable macrophage responses) should be discussed with more caution.

Thank you for the insightful comment. We have revised the conclusion to address the relevance of the assays.

We have explained how these preliminary assays were designed to assess key macrophage responses, such as activation markers and cytokine profiles, which are critical for evaluating potential therapeutic strategies. We have acknowledged the limitations of these initial tests and emphasize that while they provide valuable insights, further experiments are needed to fully understand the therapeutic potential. We also outline Future Directions on how these results guide the design of more detailed studies, focusing on specific therapeutic outcomes and their translation to in vivo models.

Here is the newly incorporated text in the conclusion

“Summarizing, we investigated ATO NPs as a potential photothermal tool in anticancer therapies. The desirable features of ATO NPs, such as their wavelength tunable photothermal conversion, colloidal stability in cell culture media, and biocompatibility, are matched with desirable macrophage responses and uptake mechanisms. However, further research is essential to assess their interaction with other immune components and their behavior in more complex biological systems, particularly through in vivo studies. While our in vitro data, including the absence of significant immune reactions and detailed uptake mapping via FIB-SEM, provide valuable insights, these results are preliminary. They lay the groundwork for more comprehensive in vivo research to advance ATO NPs toward clinical translation as targeted and effective anticancer agents.”

 

6. Some sentences in the text are quite difficult to follow, thinking about an audience of non experts - e.g. L37-40, L220-251. Maybe a re-reading of the whole paper could be beneficial, thinking to a potential audience interested in the biological effects.

We thank the reviewer for this comment. To make the text more accessible to a broader audience, particularly those interested in the biological effects, we have revised the the approach, simplifying the language and clarifying complex sentences in the manuscript, which have been highlighted.

7. Also the abstract could be made easier to understand.

Abstract is amended

Antimony-doped tin oxide nanoparticles (ATO NPs) have emerged as a promising tool in biomedical applications, namely robust photothermal effects upon near-infrared (NIR) light exposure, enabling controlled thermal dynamics to induce spatial cell death. This study investigated the interplay between ATO NPs and macrophages, understanding cellular uptake and cytokine release. ATO NPs demonstrated biocompatibility with no impact on macrophage viability and cytokine secretion. These findings highlight the potential of ATO NPs for inducing targeted cell death in cancer treatments, leveraging their unique NIR properties and safe interactions with immune cells. ATO NPs offer a transformative platform with significant potential for future biomedical applications by combining photothermal capabilities and biocompatibility.

Figure legends are always missing details which could make them understandable without reading the whole paper - too many abbreviations and no explanations.

The inconsistencies in figure legends were addressed.

 L89 I think that sentences should never start with abbreviations, symbols or numbers

We thank the reviewer for this comment we have addressed it in the revised manuscript.

 

8. L94 I think it is esential to express centrifugation speed in xg, unless a full description of the centrifuge dimensions and features is given

We thank the reviewer for this comment; centrifuge speeds are presented in xg throughout the paper.

 

9. L102, L109, L121 - for equipments and kits it is better to report in parentheses the model, brand name, city and country

We thank the reviewer for this comment, although we have followed the MDPI style requires providing full details of the equipment used.

 

10. L128-129, L134 please explain these concentrations - v/v from a stock solution? there is no way of understanding the quantities

L138 I think some abbreviations have not been defined

L144 pay attention to measure units – mL

We thank the reviewer for this comment we have addressed it in the revised manuscript.

11. L178 complete the indication on concentration

L183-185 details are missing here

L235 I do not see the size distribution in the figure

We thank the reviewer for this comment, which we have addressed in the revised manuscript.

12. L273-274 is there any way to show that indeed the thermal gradient is getting larger, with numerical indications?

We thank the reviewer for this comment. The figure in question has been amended to present the exact gradient values.

 

13. L281-282 and Table S3 - I am not familiar with this assay but surely there cannot be so many significant digits in these measurements? The text already reports the values, so why include the Table?

We thank the reviewer for this comment, the data was amended to present the appropriate number of significant digits.

 

14. L327 there are  no "cells" in the red boxes

We thank the reviewer for this comment and apologize for that mistake. Yes, the red boxes illustrate the selected areas of cells. The figure legend now addresses this.

 

15. L335 how can the authors demonstrate that these are indeed the ATO NPs? some identification should be possible with EDX analysis as in Figure S2? Figure 4B is not clear about what can be seen and identified as NPs

Nanoparticles are too small to be visualized by the instrument used. EDX analyses were presented as Fig S2B. The coupling of Sb/Sn signals and the oxygen/carbon ratio enhancement indirectly proves the presence of ATO NPs within the specific cell areas.

 

16. L352-357 highly speculative; no changes were evidenced in Figure 2A

L384 a part of Figure S3B is not visible in the supplementary file

We thank the reviewer for this comment; we apologize for that error during the text templating. It is addressed in the revised figure.

 

17. L411 "professional phagocytes" cannot be accepted

L422-424 I do no think this has been shown in the paper

L426-428 rephrase - the metabolic activity is shown by cells, not by NPs

 We thank the reviewer for this comment, professional phagocytes have been removed and  revised with this text: “In this study, neither activated nor non-activated ATO nanoparticles (NPs) caused a sub-stantial decrease in the metabolic activity exhibited by J774A.1 cells at concentrations up to 100 μg/mL”

 

Comments on the Quality of English Language

The English language is very good - nonetheless some parts of the paper are very difficult to read thinking to a potential audience interested in "cells"

Reviewer 2 Report

Comments and Suggestions for Authors

In this manuscript, Olexiy et al delivered a study on the interaction of ATO NPs with macrophages, focusing on its NPs uptake and cytokine release properties. Chemical synthesis, characterizations, and biological activity tests are well described and presented in the manuscript. This is an interesting work and it's recommended for publication after some minor revisions.

1. Dynamic light scattering test should be provided for showing the size distribution of NPs.

2. Optical properties of NPs, such as light absorption spectrum, should be provided to show the wavelength of absorption in NIR region.

3. ATP NPs was used for photothermal treatment of J774A.1, but how about its photothermal conversion efficiency?

Author Response

In this manuscript, Olexiy et al delivered a study on the interaction of ATO NPs with macrophages, focusing on its NPs uptake and cytokine release properties. Chemical synthesis, characterizations, and biological activity tests are well described and presented in the manuscript. This is an interesting work and it's recommended for publication after some minor revisions.

1. Dynamic light scattering test should be provided for showing the size distribution of NPs.

We thank the reviewer for this comment. In Supplementary table S1, we provide the data extracted from the DLS distribution. The DLS histogram would then add no additional information to the already tabulated data.

Table S1. DLS measured Zeta potential and hydrodynamic diameter distributions of ATO NPs in Milli-Q water.

ATO NP concentration, μg/mL	Dh, nm	PDI	z. meV
25	46± 2	0.184	-31.0±1.3
100	44± 1	0.141	-32.3±1.9

 

2. Optical properties of NPs, such as light absorption spectrum, should be provided to show the wavelength of absorption in NIR region.

We thank the reviewer for this comment, in Supplementary we provide the UV-vis-NIR absorbances (fig. S1A and B) of both undoped TO and ATO nanoparticles. For ATO NPs we located the position of LSPR peak.

3. ATO NPs was used for photothermal treatment of J774A.1, but how about its photothermal conversion efficiency?

Thank you for your insightful question regarding the photothermal conversion efficiency of ATO NPs. In this study, our primary focus was establishing a proof of concept through qualitative assessments rather than quantitative analysis. We employed lock-in thermography, which is highly sensitive and well-suited for qualitative elucidation of thermoconversion, particularly at low power. However, due to the constraints of our PPTT setup, which was not equipped with an infrared (IR) camera, we could not perform real-time thermal mapping within the well plate. This limitation prevented us from acquiring the quantitative figures of merit typically used to determine photothermal conversion efficiency.

 

We acknowledge the importance of these quantitative metrics and are considering future studies with enhanced equipment that would allow us to provide these figures. Nonetheless, the current qualitative results strongly support the potential of ATO NPs for photothermal treatment, as evidenced by the proof of concept presented in our work.

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript “ Understanding Macrophage Interaction with Antimony-Doped 2 Tin Oxide Plasmonic Nanoparticles” is well written; however, the conclusion cannot be drawn from the present study that ATO NPs can be used as PTT agents.  The findings strongly indicated the need for future research such as optimum conditions when ATO NPs can be used as PTT agent under NIR-II lasers.  In my opinion, the manuscript is not suitable for publication at this stage. I have following concerns which need to be addressed.  

1.      Can the authors calculate the photothermal efficiency of ATO NPs using laser power of 10W/cm²  which shows decrease in cell viability of J774A.1 macrophage in FigS3?

2.      Did the authors attempt a longer irradiation duration using laser (1064 nm) of power (1W/cm² or 5W/cm²), such as 5 min, or 10 min to confirm the photo-induced toxicity of J774A.1 macrophage.

3.      Please provide the complete figure Fig S3 (B) which is not visible properly. The significant toxicity of J774A.1 macrophage in controls (untreated + NIR laser 1064nm) in Fig S3 raises a concern about the damage to healthy cells.

4.      LIT thermal maps showed higher thermal response of ATO at higher conc. Of  100ug/mL using laser (940nm) in figure 3A. However, this concentration-dependent heating effect is not visible in Fig S3A, B when higher laser of 1064 nm with a power of 10W/cm² was used. Please justify.

Author Response

The manuscript “ Understanding Macrophage Interaction with Antimony-Doped 2 Tin Oxide Plasmonic Nanoparticles” is well written; however, the conclusion cannot be drawn from the present study that ATO NPs can be used as PTT agents.  The findings strongly indicated the need for future research such as optimum conditions when ATO NPs can be used as PTT agent under NIR-II lasers.  In my opinion, the manuscript is not suitable for publication at this stage. I have following concerns which need to be addressed. 

Thank you for your feedback. We acknowledge that our study provides foundational insights rather than conclusive evidence that ATO NPs can be used as PPTT agents. We have revised the manuscript to emphasize the need for further research to optimize the conditions under which ATO NPs can be used effectively, particularly with NIR-II lasers. We believe these revisions address your concerns and clarify the scope of our findings.

 

1. Can the authors calculate the photothermal efficiency of ATO NPs using laser power of 10W/cm2 which shows decrease in cell viability of J774A.1 macrophage in FigS3?

Thank you for your insightful question regarding the photothermal conversion efficiency of ATO NPs. In this study, our primary focus was on establishing a proof of concept through qualitative assessments rather than quantitative analysis. We employed lock-in thermography, which is highly sensitive and well-suited for qualitative elucidation of thermoconversion, particularly at low power. However, due to the constraints of our PPTT setup, which was not equipped with an infrared (IR) camera, we were unable to perform real-time thermal mapping within the well plate. This limitation prevented us from acquiring the quantitative figures of merit typically used to determine photothermal conversion efficiency.

We acknowledge the importance of these quantitative metrics and are considering future studies with enhanced equipment that would allow us to provide these figures. Nonetheless, the current qualitative results strongly support the potential of ATO NPs for photothermal treatment, as evidenced by the proof of concept presented in our work.

2. Did the authors attempt a longer irradiation duration using laser (1064 nm) of power (1W/cm2 or 5W/cm2), such as 5 min, or 10 min to confirm the photo-induced toxicity of J774A.1 macrophage.

Thank you for your insightful suggestion. Our current study focused on evaluating the initial photothermal effects and biocompatibility of ATO NPs with relatively short irradiation durations. However, we did not explore longer irradiation times, such as 5 or 10 minutes, using a 1064 nm laser at 1W/cm² or 5W/cm². We agree that extending the irradiation duration could provide valuable insights into the potential photo-induced toxicity and enhance our understanding of the therapeutic window. We will consider incorporating this in future experiments to confirm further and optimize ATO NP-mediated photothermal therapy conditions.

3. Please provide the complete figure Fig S3 (B) which is not visible properly. The significant toxicity of J774A.1 macrophage in controls (untreated + NIR laser 1064nm) in Fig S3 raises a concern about the damage to healthy cells.

We apologize for the incomplete presentation of Fig. S3(B). It was probably not visible when it was converted to PDF. To ensure clarity, the figure will be revised and presented in full detail in the updated version of the manuscript.

 

Regarding your concern about the significant toxicity observed in the controls (untreated + NIR laser 1064 nm), we acknowledge this is an important issue to address. The observed toxicity in control cells suggests that prolonged exposure to the NIR laser alone can induce cellular damage, potentially impacting healthy tissues in a therapeutic context. This highlights the need to carefully optimize laser parameters, including power density and irradiation time, to minimize unintended damage to healthy cells during photothermal therapy.

In future experiments, we explore these parameters further to refine the balance between effective cancer cell targeting and minimizing collateral damage to healthy cells. Additionally, we will include a more thorough discussion of these findings in the revised manuscript to address this concern adequately.

4. LIT thermal maps showed higher thermal response of ATO at higher conc. Of 100ug/mL using laser (940nm) in figure 3A. However, this concentration-dependent heating effect is not visible in Fig S3A, B when higher laser of 1064 nm with a power of 10W/cm2 was used. Please justify.

Thank you for pointing out this observation. The discrepancy between the thermal response observed at higher concentrations of ATO nanoparticles (100 µg/mL) using a 940 nm LED in Figure 3A and the lack of a similar concentration-dependent heating effect when using a 1064 nm laser at 10 W/cm² in Fig S3A and B requires careful consideration.

This difference can be attributed to the distinct excitation dynamics of the ATO nanoparticles within the two different methods (LIT and PPTT), despite the wavelength used (940 nm and 1064 nm, respectively) are roughly within same proximity to the peak of ATO NPs absorption spectrum. As a modulated (pulsed) probe, LIT balances low-intensity LED photothermal heating and heat dissipation; as a result, the internalized nanoparticles exhibit a more pronounced photothermal response, particularly at higher concentrations, as seen in Figure 3A. The LIT is an add-on reverifying the uptake scenario for ATO nanoparticles.

In contrast, when exposed to the 1064 nm laser at a much higher power density of 10 W/cm², the non-linear effects might contribute to different thermal behavior, where the nanoparticles’ heating/ heat dissipation does not scale linearly with concentration; moreover in the case wherein laser can solely overcome the hyperthermia level. This could explain the absence of an apparent concentration-dependent viability effect in Fig S3A and B.

We have included this justification in the revised manuscript and expanded the discussion to further elucidate the differences in ATO nanoparticles' photothermal behavior under varying NIR excitation conditions.

Here is the newly incorporated text in the discussion

However, when different light sources and power densities are employed, addressing the observed discrepancies in photothermal responses is vital. The decent thermal response differences between J774A.1 cells exposed for 24 h at 25 μg/mL and 100 μg/mL of ATO NPs under 940 nm irradiation (Figure 3A) does not appear as pronounced when a 1064 nm laser at 10 W/cm² is used (Fig S3A, B). This discrepancy can be attributed to the substantial readout differences for the LIT and PPTT probes. The former is an ultrasensitive modulation technique capable of resolving ultrasmall thermal gradients, which serves as additional evidence of the ATO NPs uptake. The PPTT generates less pronounced concentration dependencies in thermal effect as it may solely overcome the cells’ hyperthermia level, which is seen for the ATO untreated sample. The nonlinear effects in heat dissipation at high power density may further contribute to the observed differences, underscoring the importance of optimizing PPTT parameters, namely laser wavelength and power, duration of treatment, and the precise control of initial temperature [41] for maximizing the photothermal efficiency of ATO NPs for the targeted cells.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I thank the authors for considering my comments.

Author Response

We thank the reviewer for his/her overall positive comments on our paper.

Reviewer 3 Report

Comments and Suggestions for Authors

The author of the manuscript “Understanding Macrophage Interaction with Antimony-Doped 2 Tin Oxide Plasmonic Nanoparticles” has provided satisfactory answers to the concerns raised and therefore, suitable for publication after minor changes follows

 

1.      Ref. 21 and 44 are irrelevant and should be removed.

2.      Please mention NIR laser wavelength in Fig 5 and Fig.S3.

Author Response

We thank the reviewer for his/her overall positive comments on our paper.

The references in question were replaced, and NIR wavelength was mentioned in the legends of the figures.