From the journal RSC Chemical Biology Peer review history

26-Aug-2022

Dear Dr Ryu:

Manuscript ID: CB-ART-07-2022-000166
TITLE: Enzyme-instructed morphology transformation of mitochondria-targeting peptide for the selective eradication of osteosarcoma

Thank you for your submission to RSC Chemical Biology, published by the Royal Society of Chemistry. I sent your manuscript to reviewers and I have now received their reports which are copied below.

I have carefully evaluated your manuscript and the reviewers’ reports, and the reports indicate that major revisions are necessary. I would like to point out to address all reviewers comments in particular to highlight the novelty of your work in comparison with already reported work, and to demonstrate the reported phenotype in additional cell lines other than Saos2 cells.

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I look forward to receiving your revised manuscript.

Yours sincerely,
Professor Zaneta Nikolovska-Coleska
Associate Editor, RSC Chemical Biology

************

Reviewer 1

This work done by Jeena et al. described the alkaline phosphatase (ALP) instructed morphological transformation from micelles to nanofibers, which can selectively occur in ALP over-expressed Saos-2 cells. The intracellular nanofibers bearing TPPP formed inside mitochondria which eventually inhibited the cancer cells. This strategy showed great potential in novel osteosarcoma treatments. Overall, this manuscript is well organized and the data adequately support the conclusion. Therefore, we would suggest a minor revision before it can be accepted for publication.

1. Regarding the choice of NBD dye, we would suggest citing proper references, e.g., Nat. Commun., 2012, 3, 1033; Langmuir, 2013, 29, 15191-15200.

2. The author should double-check the molecule identities (Mito-FFYp or Mito-FFY) thoroughly. For example, on page 4, the authors wrote “… that Mito-FFYp diffuses freely inside the cell instead of…”. As described above and in the scheme, it should be Mito-FFY instead of Mito-FFYp. Similarly, in the conclusion section, “… Mito-FFYp formed fibers within the mitochondria in cancer cells…” it should be Mito-FFY.

3. This mito-targeted assembly strategy is quite attractive for osteosarcoma treatment. While the current study has shown the capability to inhibit Saos-2 cells, we would suggest a more detailed discussion on the potential in vivo study.

4. Please state the chirality of the amino acids used in this manuscript and in all the chemical structures.

Reviewer 2

The manuscript by Ryu et al. reported a mitochondria-targeting peptide Mito-FFYp for the eradication of osteosarcoma. They determined the CAC and zetapotential of the peptide, and characterized the enzyme-instructed morphology transformation of Mito-FFYp. Moreover, the cell cytotoxicity against Saos-2 cells was determined and the co-localization of Mito-FFYp and mitochondria was verified. The author found that the passive diffusion and in-situ self-assembly of Mito-FFY disrupted mitochondria to induce apoptosis. This work demonstrated the self-assembling of peptide Mito-FFYp and its mechanism in treating osteosarcoma. Therefore, I recommend acceptance of this manuscript after addressing the following issues.
1. I think the authors need to discuss the major difference of this work with the work of Wang et al. (10.1021/jacs.6b09783) in which the same TPP and similar phosphopeptide used to target the same cell line Saos2.
2. (Page 2) Please briefly explain why 15 μM solution is used for further testing.
3. (Figure1 B) Please briefly explain why the difference of zetapotential suggests easy cell internalization.
4. The authors are suggested to use more endocytosis inhibitors to fully establish the passive diffusion of Mito-FFYp.
5. (Page 4) Please mention the full name of DHE in the manuscript.
6. (Figure S7) Please explain area of different colors on the figure in the caption.
7. It would be more convincing if more cell lines with high ALP are tested for the selectivity of Mito-FFYp toward ALP other than Saos-2.
8. The authors should use Mito-FFY as a control to treat cells to confirm the role of phosphate.
9. I would suggest the authors use an inhibitor of ALP to prove the enzyme conversion is essential.

Dear Prof. Nikolovska-Coleska,

Please find the attached revised manuscript (CB-ART-07-2022-000166) entitled “Enzyme-instructed morphology transformation of mitochondria-targeting peptide for the selective eradication of osteosarcoma” which we wish to be considered for publication in the RSC Chemical Biology.

We have now addressed the issues raised by the reviewers. We have also outlined details of how we addressed each of the concerns raised by the reviewers in response-to-reviewer letter. We also revise our manuscript by arranging figures in main text and supplementary information. Please find attached the modified manuscript. I have also uploaded the highlight version of the manuscript for you to easily see the modifications we made to the manuscript.

All authors and those acknowledged have reviewed the manuscript, which has not been submitted or published in any other journal.

Thank you for your consideration and we look forward to hearing from you. Please let me know if you have any questions.

Sincerely,

Ja-Hyoung Ryu

The following response to the reviewers has been copied from the file provided by the authors, without images or special characters.

Referee: 1
Comments to the Author
This work done by Jeena et al. described the alkaline phosphatase (ALP) instructed morphological transformation from micelles to nanofibers, which can selectively occur in ALP over-expressed Saos-2 cells. The intracellular nanofibers bearing TPPP formed inside mitochondria which eventually inhibited the cancer cells. This strategy showed great potential in novel osteosarcoma treatments. Overall, this manuscript is well organized and the data adequately support the conclusion. Therefore, we would suggest a minor revision before it can be accepted for publication.

Comment 1. Regarding the choice of NBD dye, we would suggest citing proper references, e.g., Nat. Commun., 2012, 3, 1033; Langmuir, 2013, 29, 15191-15200.

Response: We appreciate the reviewer for the valuable comments. We added the reference regarding the choice of NBD dye as reviewer suggested.

Reference 33 : Y. Gao, J. Shi, D. Yuan and B. Xu, Imaging enzyme-triggered self-assembly of small molecules inside live cells, Nature communications, 2012, 3, 1033
Reference 34 : Y. Gao, Y. Kuang, X. Du, J. Zhou, P. Chandran, F. Horkay and B. Xu, Imaging Self-Assembly Dependent Spatial Distribution of Small Molecules in a Cellular Environment, Langmuir, 2013, 29, 49, 15191-15200.


Comment 2. The author should double-check the molecule identities (Mito-FFYp or Mito-FFY) thoroughly. For example, on page 4, the authors wrote “… that Mito-FFYp diffuses freely inside the cell instead of…”. As described above and in the scheme, it should be Mito-FFY instead of Mito-FFYp. Similarly, in the conclusion section, “… Mito-FFYp formed fibers within the mitochondria in cancer cells…” it should be Mito-FFY.

Response: We appreciate the reviewer for the valuable comments. And we apologize for the mistake on the molecule identities. On Page 4, 2nd column, line 1, “Mito-FFYp” was revised to “Mito-FFY”. And on Page 5, conclusion, line 17, “Mito-FFY” was changed to “Mito-FFYp”.


 
Comment 3. This mito-targeted assembly strategy is quite attractive for osteosarcoma treatment. While the current study has shown the capability to inhibit Saos-2 cells, we would suggest a more detailed discussion on the potential in vivo study.

Response: We appreciate the reviewer for the valuable comments. As reviewer’s comment, we described the potential in vivo study on conclusion section. (Page 5, Line 28-32)
“Since the aggregate has higher stability in blood than the peptide monomer, we expect that Mito-FFYp micelle will have a great potential of the anticancer therapy at in vivo study on the osteosarcoma tumor composed of cancer cells with high ALP expression levels.”


Comment 4. Please state the chirality of the amino acids used in this manuscript and in all the chemical structures.

Response: We appreciate the reviewer for the valuable comments. We stated our group’s previously designed molecule, Mito-FF and synthesized compounds for this article Mito-FFYp and Mito-FFY were L-form. And we also revised the scheme to represent designed Mito-FFYp was L-form peptide.

Scheme 1. Schematic illustration of the enzyme-instructed morphology transformation (EIMT) of an ALP-responsive micelle. The ALP enzyme induces the transformation of L-Mito-FFYp micelles to positively charged Mito-FFY molecules, which enter the cell via free diffusion to target the mitochondria. Inside the mitochondria, L-Mito-FFYp self-assembles into nanofibers to induce mitochondrial dysfunction leading to cell death. Chemical structure of L-Mito-FFYp and L-Mito-FFY represented under the scheme


Referee: 2
Comments to the Author
The manuscript by Ryu et al. reported a mitochondria-targeting peptide Mito-FFYp for the eradication of osteosarcoma. They determined the CAC and zetapotential of the peptide, and characterized the enzyme-instructed morphology transformation of Mito-FFYp. Moreover, the cell cytotoxicity against Saos-2 cells was determined and the co-localization of Mito-FFYp and mitochondria was verified. The author found that the passive diffusion and in-situ self-assembly of Mito-FFY disrupted mitochondria to induce apoptosis. This work demonstrated the self-assembling of peptide Mito-FFYp and its mechanism in treating osteosarcoma. Therefore, I recommend acceptance of this manuscript after addressing the following issues.

Comment 1. I think the authors need to discuss the major difference of this work with the work of Wang et al. (10.1021/jacs.6b09783) in which the same TPP and similar phosphopeptide used to target the same cell line Saos2.

Response: We appreciate the reviewer for the valuable comments. The reviewer mentioned article above reported on the phenomenon that the peptide monomer was cleaved by the ALP enzyme on the cell surface, and formed a self-assembled structure, and entered the cell through endocytosis. The most different major point from this article is whether the self-assembly is formed before entering the cell after the cleavage of phosphate by ALP, or the disassembly is occurred by ALP and the self-assembly is occurred in the mitochondria. To achieve self-assembly on extracellular space, the amount of molecule must be very large to reach a concentration above Critical Aggregation Concentration (CAC). However, as in this article, if the space where self-assembly occurs was confined to the mitochondria, a small amount of peptide is sufficient to reach the CAC. Therefore, the treatment efficiency can be maximized only by treating a small amount. We described the above at the end of introduction. (Page 2, 1st column, line 2-6)
In addition, when internalized in an assembled state through endocytosis, this process requires energy. However, in this paper, the assembled structure was disassembled by ALP enzyme and the peptide monomer is passively diffused into the cell. We confirmed that when L-Mito-FFYp was treated at 4°C, it was accumulated in the mitochondria as at 37°C (Figure S7), inducing damage also. (Figure S8) That is, the L-Mito-FFYp peptide in this article is different in that it permeates into cells through passive diffusion, an energy-independent pathway. We described the above at Page 4, 1st column, line 14-17.



 

Figure S7. Confocal images showing cellular uptake at 4 ℃ condition and in presence of the clathrin-mediated endocytosis inhibitor, pitstop2.

Figure S8. Mitochondrial dysfunction at Saos-2 cell induced by L-Mito-FFYp which is measured by labeling with TMRM. L-Mito-FFYp compound was treated at 4℃ or co-incubated with pitstop2.

 
Comment 2. (Page 2) Please briefly explain why 15 μM solution is used for further testing.

Response: We appreciate the reviewer for the valuable comments. As shown in Figure 1A, the CAC of Mito-FFYp is 12 μM. And like in the scheme, the hypothesis can be verified only when the peptide maintains a state of forming a micelle outside the cell. Therefore, it was treated with 15 μM (higher than the CAC of Mito-FFYp peptide), which was converted into monomer by the EIMT process and entered the cell. In reflection of the reviewer’s comments, the sentence “over the CAC value” was added to this article (Page 2, 2nd column, Line 3)

Figure 1. A) CAC determination of Mito-FFYp using pyrene excitation method.

Scheme 1. Schematic illustration of the enzyme-instructed morphology transformation (EIMT) of an ALP-responsive micelle. The ALP enzyme induces the transformation of L-Mito-FFYp micelles to positively charged Mito-FFY molecules, which enter the cell via free diffusion to target the mitochondria. Inside the mitochondria, L-Mito-FFYp self-assembles into nanofibers to induce mitochondrial dysfunction leading to cell death. Chemical structure of L-Mito-FFYp and L-Mito-FFY represented under the scheme.
Comment 3. (Figure 1B) Please briefly explain why the difference of zetapotential suggests easy cell internalization.

Response: We appreciate the reviewer for the valuable comments. Cell membrane potential has a negative value, as can be confirmed in the following paper. Therefore, Mito-FFYp micelles with a surface charge of -25.6 mV are difficult to penetrate cells, but Mito-FFY with positive charges can penetrate more effectively into cells. The conversion of Mito-FFYp to Mito-FFY by ALP occurs much more readily in ALP-overexpressed Saos-2, and thus has selective toxicity to cancer cells. We described the above at Page 2, 2nd column, line 6.


Reference 32 : M. Yang and W. J. Brackenbury, Membrane potential and cancer progression, Frontiers in Physiology, 2013, 4, 185.



 
Comment 4. The authors are suggested to use more endocytosis inhibitors to fully establish the passive diffusion of Mito-FFYp.

Response: We appreciate the reviewer for the valuable comments. We treated Mito-FFYp with another clathrin-mediated endocytosis inhibitor, pitstop2. Despite in presence of the endocytosis inhibitor, it accumulated in the Saos-2 cell mitochondria (Figure S7) and it induced damaged to mitochondria (Figure S8). Also, cellular uptake at 4 ℃ showed similar uptake as 37 ℃. It suggests the Mito-FFYp peptides changed into Mito-FFY and Mito-FFY internalized to the cell through energy independent pathway. We described the above at Page 4, 1st column, line 14-22.


Figure S7. Confocal images showing cellular uptake at 4 ℃ condition and in presence of the clathrin-mediated endocytosis inhibitor, pitstop2.

Figure S8. Mitochondrial dysfunction at Saos-2 cell induced by Mito-FFYp which is measured by labeling with TMRM.
Comment 5. (Page 4) Please mention the full name of DHE in the manuscript.

Response: We appreciate the reviewer for the valuable comments. On page 4, 1st column, line 32, we mention the full name of DHE in the manuscript. “In addition, staining with dihydroethidium (DHE) produced a deep red fluorescence….”



Comment 6. (Figure S7) Please explain area of different colors on the figure in the caption.

Response: We appreciate the reviewer for the valuable comments. And we apologize for not having detailed descriptions of each color band in original Figure S7. (Not it was changed to Figure S9.) We added explanation on Figure S9.


Figure S9. Corresponding flow cytometric analysis of Figure 2E, F.

 
Comment 7. It would be more convincing if more cell lines with high ALP are tested for the selectivity of Mito-FFYp toward ALP other than Saos-2.

Response: We appreciate the reviewer for the valuable comments. In the following references, it was confirmed that the HeLa cancer cell line was selected as an ALP overexpressed cancer cell and tested. Therefore, in this paper, a toxicity test of Mito-FFYp on HeLa cells was conducted, and showed high toxicity similar to Saos-2 cells. These results were updated by putting them in Figure 3A.

References
[37] Z. Zhou, X. Du, C. Berciu, H. He, J. Shi, D. Nicastro and B. Xu, Enzyme-Instructed Self-Assembly for Spatiotemporal Profiling of the Activities of Alkaline Phosphatases on Live Cells, 2016, Chem, 1, 246-263.
[38] W. Tan, Q. Zhang, M. C. Q. -Frias, A. Y. Hsu, Y. Zhang, A. Rodal, P. Hong, H. R. Luo, B. Xu, Enzyme-Responsive Peptide Thioesters for Targeting Golgi Apparatus, J. Am. Che. Soc. 2022, 144m 6709-6713.

Figure 3. A) Cell viability analysis of Mito-FFYp in Saos-2, HeLa, SK-BR-3, and NIH 3T3 cell lines for an incubation time of 48 h.

 
Comment 8. The authors should use Mito-FFY as a control to treat cells to confirm the role of phosphate.

Response: We appreciate the reviewer for the valuable comments. We synthesized and purified Mito-FFY compound to verify the important role of phosphate. (Figure S2). It was supposed after the phosphate group cleaved from the ALP enzyme. The L-tyrosine phosphate molecule was replaced to L-tyrosine. Mito-FFY also showed high accumulation into the mitochondria (Figure S10) and it induced damage to the mitochondria (Figure S11) as same as Mito-FFYp. However, different from Mito-FFYp, Mito-FFY compound made the loss of mitochondrial integrity on healthy NIH-3T3 cell also (Figure S12). And cytotoxicity was highly increased at NIH-3T3 cell after 48 h incubation with Mito-FFY (Figure S13). Therefore, from these results, phosphate group grants more selectivity through EIMT process. We described the above at (page 4, 2nd column, line 15 – 22).


Figure S2. A) Chemical structure of Mito-FFY, B) MALDI-TOF spectra of Mito-FFY after HPLC purification that confirms the successful formation of the product, C) TEM images of Mito-FFY showing the presence of nanofibers of diameter ~ 10 nm.


Figure S10. Confocal images showing cellular uptake of Mito-FFY at Saos-2 cell.




Figure S11. Mitochondrial dysfunction at Saos-2 cell induced by Mito-FFY which is measured by labeling with TMRM.




Figure S12. Mitochondrial dysfunction at NIH-3T3 cell induced by Mito-FFY which is measured by labeling with TMRM.


Figure S13. Cell viability analysis of Mito-FFY in Saos-2 and NIH 3T3 cell lines for an incubation time of 48 h.

 
Comment 9. I would suggest the authors use an inhibitor of ALP to prove the enzyme conversion is essential.

Response: We appreciate the reviewer for the valuable comments. We purchased a Nitro-blue tetrazolium chloride/5-bromo-4-chloro-3’-indolyphosphate p-toluidine salt (NBT-BCIP) solution known as an ALP inhibitor from Sigma-Aldrich to confirm the importance of enzyme conversion. First, the change in self-assembled morphology after inhibitor treatment was observed. (Figure R1) When Mito-FFYp was treated with enzyme and inhibitor simultaneously, micelles similar as Mito-FFYp were observed, unlike Figure. 1D. And we compared the toxicity of Mito-FFYp at Saos-2 cell between absence of the inhibitor and pre-incubation with the inhibitor. As a result, it was observed that the toxicity was highly reduced. (Figure S14) Above explanation was described at page 4, 2nd column, line 22 – page 5, 1st column, line 3.


Figure R1. TEM image of Mito-FFYp and Mito-FFYp + ALP + ALP inhibitor (NBT-BCIP).



Figure 1. D) TEM analysis of 15 μM of Mito-FFYp + ALP.



Figure S14. Cell viability analysis of Mito-FFYp in the presence of ALP inhibitor, NBT-BCIP.

10-Oct-2022

Dear Dr Ryu:

Manuscript ID: CB-ART-07-2022-000166.R1
TITLE: Enzyme-instructed morphology transformation of mitochondria-targeting peptide for the selective eradication of osteosarcoma

Thank you for submitting your revised manuscript to RSC Chemical Biology. I am pleased to accept your manuscript for publication in its current form. I have copied any final comments from the reviewer(s) below.

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Reviewer 1

The authors have addressed all the comments in full. Therefore, we would suggest the acceptance of this manuscript in its current version.

Reviewer 2

The authors have addressed my comments, and I support the acceptance of this work.