From the journal RSC Chemical Biology Peer review history
A ruthenium–oligonucleotide bioconjugated photosensitizing aptamer for cancer cell specific photodynamic therapy
Round 1
Manuscript submitted on 13 Qad 2021![](https://www.rsc-cdn.org/pubs-core/2022.0.147/content/NewImages/icon-letter.png)
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13-Sep-2021
Dear Dr Hollenstein:
Manuscript ID: CB-ART-07-2021-000146
TITLE: A ruthenium-oligonucleotide bioconjugated photosensitizing aptamer for cancer cell specific photodynamic therapy
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Yours sincerely,
Claudia Höbartner
Associate Editor, RSC Chemical Biology
Institute of Organic Chemistry, University of Würzburg
************
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Reviewer 1
The team of Gilles Gasser and Marcel Hollenstein report in their paper titled “A ruthenium-oligonucleotide bioconjugated photosensitizing aptamer for cancer cell specific photodynamic therapy“ on a series of conjugates of Ru-complexes with nucleolin-targeting AS1411 aptamer. My comments:
- Fig. S4: 1H NMR spectrum of RuN3 complex shows few impurity peaks: for example, between 3.75 and 4.00 ppm; between 2.65 and 3.00 ppm. The spectrum was cut at 1 ppm. It would make sense to show it starting from 0 ppm. It would be desirable to confirm the purity of RuN3 by elemental analysis or, if the amount available is too small, by LC-MS.
- Fig. S16: LC-MS traces of Ru-complex~aptamer conjugates show several peaks: a major one and several later eluted shoulders. Are the conjugates pure? The authors should clarify this issue.
- Fig. S19: The authors use the data in this figure as an evidence of enhanced uptake of the Ru-complex~aptamer conjugates into cancer cells versus normal cells. Without accurate signal quantification and normalization, this conclusion can not be made from the data shown. I have my doubts since the blank signal in case of MCF-7 cells is much stronger than that in case of RPE-cells. This might indicate that the different threshholds were applied that might lead to the misleading final result.
- Fig. S20: This is a key result in the paper. The authors did not provide statistical treatment of the data: for example Student’s t test or any related test. Since the standard deviations are very high, the conclusions discussed in the paper are not accurately supported without the statistical analysis.
If the effect is present (the stronger killing of cancer cells vs normal cells) than only at one concentration of the conjugate and one incubation time. How the authors explain this fact. Some discussion should be provided.
- Why the cancer cell specificity is so low despite the fact that the cancer cells used by the authors express so much more of the correponding receptor than the normal cells used? Some comments from the authors would be welcome.
Conclusion: If the authors address the issues mentioned above, the paper may become suitable for publication.
Reviewer 2
EVALUATION:
Reviewer's Responses to Questions
1. Suitability of the article for the journal’s scope?
Well suited
2. Impact and novelty of the work?
Highly relevant for future PDT development
3. Length of the article - does it reflect the level of scientific content and fit within any relevant page limits?
Appropriate
4. Whether the article type is appropriate?
Yes
5. The title - does it reflect the content and contain relevant search terms for discoverability?
Yes
6. The abstract - is it self-contained without reference to the main text?
Yes
7. Which revisions are major concerns preventing publication, and which are minor concerns the authors can easily resolve, and indicate this in your report.
__________________________________
Reviewer report and recommendation
The full article ‘A ruthenium-oligonucleotide bioconjugated photosensitizing aptamer for cancer cell specific photodynamic therapy’ submitted by Hollenstein, Gasser and co-workers reports on the cancer cell specific targeting strategy based on the use of bioconjugated anti-nucleolin aptamer AS1411 that, via click-chemistry, has been used for the first time in direct covalent conjugation of the aptamer to a known, highly potent Ru polypyridyl singlet oxygen photosensitizer. A series of PS differing in either the length of the nucleobase spacers or the position of AS1411 functionalization (3’/5’) was obtained, with adequate structural, photophysical as well as a thorough biophysical characterization of the new compounds given.
Although the content of the manuscript is yet not related to Xeno-nucleic acids, publication in the themed collection can be considered upon minor revisions, as the new strategy given can be expanded to other systems using XNA aptamers in future, making the article suitable for the journal’s scope within the collection. The impact of the scientific contribution is considered to be highly relevant for the readership of RSC Chemical Biology. It’s length and submission as a full article is appropriate, with the necessary details given in the ESI. The title reflects the content and provides relevant search terms for discoverability. The abstract is self-contained and arouses the reader’s curiosity. All relevant previous literature has been cited by the authors. The challenges faced are well discussed, giving insight into ‘negative results’ too. Biological experiments have been performed under consistent conditions compared to those carried out with the model compound without aptamer functionalization to allow for comparison of the data. Nuclease stability tests as well as G-quadruplex formation gave important insight into the recommended conjugation site of the aptamer.
Reviewer comments to the authors
(1) In the section of introduction, the ability of G-rich oligonucleotides like aptamer AS1411 to form a G-quadruplex in the presence of certain monovalent metal cations like K+ is described. The data shown in the manuscript seems to support the hypothesis that G-quadruplex formation might be required for cellular uptake. As CD spectroscopy, thermal difference spectra and Tm melting experiments were used to probe the ability, are more details known about the mechanistics of cellular uptake?
(2) Table 2 lists the spectroscopic properties for RuN3 - however, UV/Vis absorption properties are missing here. Also, for better comparability of phototoxicity data with [Ru(Bphen)2(4,4’ dmbpy)]2+, molar absorption coefficients of the (well soluble) compounds would be nice. For Ru-AS1411 complexes this might not be possible due to only partial solubility upon drying by speed vac.
(3) Graphical representation of the LD50 values under given experimental conditions might be more vivid than presenting all data in tables (similar to Figures S20).
(4) In the experimental section, samples prepared for lifetime measurements (as well as singlet oxygen yield determination) are described to have an optical density of 0.2 - wouldn’t it be more appropriate to do such measurements with solutions of 0.1 OD as well, due to inner filter effects?
(5) Figure S10 - The IR spectrum of RuN3 is shown; typically, azide stretching bands are very strong in intensity - why is it so weak here?
Minor comments:
The chemical stability of RuN3 should be checked carefully as its application in living cells is discussed on page 11 and 12. Azides are known to be very reactive.
The access to the azide containing ruthenium complex appears to be very relevant to several fields of application of such complexes. This fact should be highlighted in the introduction appropriately. See for instance Zabarska et al. Dalton Trans., 2016,45, 2338-2351.
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October 6, 2021
Professor Claudia Höbartner
Associate Editor,
RSC Chemical Biology
Dear Professor Höbartner,
Thank you for the positive assessment of our manuscript and the excellent comments made by the reviewers. Hereby, we would like to submit a revised version of our manuscript entitled “A ruthenium-oligonucleotide bioconjugated photosensitizing aptamer for cancer cell specific photodynamic therapy”, by Luke K. McKenzie, Marie Flamme, Patrick S. Felder, Johannes Karges, Frederic Bonhomme, Albert Gandioso, Christian Malosse, Gilles Gasser*, and Marcel Hollenstein* (CB-ART-07-2021-000146).
Please find attached a point-to-point response to the corrections and comments of the referees along with the revised manuscript and supporting information.
We hope we could answer all the concerns raised by the referees, especially concerning the integrity of the Ruthenium azide complex and the statistical analysis. We have now included additional figures in the supporting information and changed the manuscript substantially.
Yours Sincerely,
Dr. Marcel Hollenstein
Referee 1
- Fig. S4: 1H NMR spectrum of RuN3 complex shows few impurity peaks: for example, between 3.75 and 4.00 ppm; between 2.65 and 3.00 ppm. The spectrum was cut at 1 ppm. It would make sense to show it starting from 0 ppm. It would be desirable to confirm the purity of RuN3 by elemental analysis or, if the amount available is too small, by LC-MS.
We thank the reviewer for their comment. We have now included an NMR of the sample used in the biological assays starting from 0 ppm. As can be seen in this sample we do not have the same trace impurities seen before. In addition, our HRMS data clearly confirms the integrity of the RuN3. This is also indirectly confirmed by the successful click reactions on the alkyne-modified AS1411 sequences. Lastly, LCMS analysis of these types of compounds is not a trivial undertaking which often results on broad peaks in LC.
- Fig. S16: LC-MS traces of Ru-complex~aptamer conjugates show several peaks: a major one and several later eluted shoulders. Are the conjugates pure? The authors should clarify this issue.
We thank the referee for their comment. In our experience, these shoulders appear frequently in the LC-MS of oligonucleotides and we believe they are due to differing conformational arrangements of the oligonucleotides. This can certainly be the case since AS1411 adopts a G4-like structure. The mass measurement of the eluted shoulders correspond to the mass of the aptamer conjugates. We have added clarification in the SI to state ‘EM=10072 (tr = 5.84) (equivalent mass at tr = 5.93)’ and ‘EM = 10560 (tr = 5.87 min) (equivalent mass at tr = 5.96, tr = 6.04)’ for AS1411-5’-TT-Ru and AS1411-3’-TTT-Ru respectively. In addition to this, PAGE gels of the Ru-aptamers show one band, as seen for the 0 time points in Figure S21, further indicating purity of the oligonucleotides.
- Fig. S19: The authors use the data in this figure as an evidence of enhanced uptake of the Ru-complex~aptamer conjugates into cancer cells versus normal cells. Without accurate signal quantification and normalization, this conclusion can not be made from the data shown. I have my doubts since the blank signal in case of MCF-7 cells is much stronger than that in case of RPE-cells. This might indicate that the different threshholds were applied that might lead to the misleading final result.
We thank the reviewer for their comment. All samples were imaged using the same exact microscope settings with no changes to gain applied post imaging. The difference in signal between blank MCF-7 and RPE-1 cells is due to differing background auto-fluorescence rather than image processing. Nonetheless, we have removed reference to enhanced uptake in relation to the microscopy data. The sentence referencing the imaging data in the main text now reads – ‘Confocal microscopy confirmed uptake in MCF7 and RPE-1 cells (Figure S19)’
- Fig. S20: This is a key result in the paper. The authors did not provide statistical treatment of the data: for example Student’s t test or any related test. Since the standard deviations are very high, the conclusions discussed in the paper are not accurately supported without the statistical analysis.
If the effect is present (the stronger killing of cancer cells vs normal cells) than only at one concentration of the conjugate and one incubation time. How the authors explain this fact. Some discussion should be provided.
We thank the reviewer for their comment and fully agree; this was an oversight on our part. We have performed a student t test on the data concerned resulting in a value of 0.00798. As such we have indicated the significance on the graph with ** and specified the significance in the legend. We have created a new figure in the main text to highlight these data. As discussed in the main text, we believe that only this conjugate is achieving specific uptake in the target cells, and may be a result from formation of differing G-Quadruplex formation by the other two conjugates (as indicated by their Tm values) while non-specific uptake is dominating the toxicity of the other conjugates. We have added the following sentence to the main text for clarification:
‘These data may indicate that only AS1411-5’-TTTTT-Ru achieves specific uptake in the target cells, while formation of differing G-Quadruplex structures by AS1411-5’-TT-Ru and AS1411-3’-TTT-Ru (as indicated by their Tm values) may result in only non-specific uptake.‘
- Why the cancer cell specificity is so low despite the fact that the cancer cells used by the authors express so much more of the corresponding receptor than the normal cells used? Some comments from the authors would be welcome.
We thank the reviewer for their comment. We have re-reviewed the literature; unfortunately, many papers inexplicably do not use a control cell line (then go straight into mice!). However, we have found another paper with this non-specific effect:
https://pubs.acs.org/doi/pdf/10.1021/nn901374b
As discussed in the discussion section, we originally thought the non-specific uptake may be due to sample degradation in the cell medium, however the Ru-modification appears to reduce this compared to the natural oligonucleotide. We believe the increased lipophilicity imparted by the added Ruthenium may increase non-specific binding alongside non-specific uptake as seen in the paper referenced. We have added the following sentence to the main text including reference to the paper mentioned above:
‘Conjugation of the RuN3 is likely to increase the lipophilicity of the Ru-AS1411s and may increase their non-specific uptake. Non-specific uptake and photodynamic effect have been previously reported with AS1411. [ref]’
Referee: 2
Reviewer comments to the authors
(1) In the section of introduction, the ability of G-rich oligonucleotides like aptamer AS1411 to form a G-quadruplex in the presence of certain monovalent metal cations like K+ is described. The data shown in the manuscript seems to support the hypothesis that G-quadruplex formation might be required for cellular uptake. As CD spectroscopy, thermal difference spectra and Tm melting experiments were used to probe the ability, are more details known about the mechanistics of cellular uptake?
From our understanding, the pathway into the cell is through binding to the nucleolin which is abnormally expressed on the cell surface in a number of cancer cell lines. This abnormally expressed nucleolin cycles back into the cell and transports with it any bound aptamer. We were not about to find any in depth structural analysis, such as X-ray crystallography, in the literature. We have found a paper (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2970734/) suggesting that AS1411 enters cells via a different uptake mechanism:
[IMAGE]
Unfortunately a more in depth experimental study is beyond the scope of the project.
(2) Table 2 lists the spectroscopic properties for RuN3 - however, UV/Vis absorption properties are missing here. Also, for better comparability of phototoxicity data with [Ru(Bphen)2(4,4’ dmbpy)]2+, molar absorption coefficients of the (well soluble) compounds would be nice. For Ru-AS1411 complexes, this might not be possible due to only partial solubility upon drying by speed vac.
We thank the reviewer for noticing this omission; we have now added the relevant data
(3) Graphical representation of the LD50 values under given experimental conditions might be more vivid than presenting all data in tables (similar to Figures S20).
We thank the reviewer for their suggestion and we have now added a figure to the main text highlighting the most important data.
(4) In the experimental section, samples prepared for lifetime measurements (as well as singlet oxygen yield determination) are described to have an optical density of 0.2 - wouldn’t it be more appropriate to do such measurements with solutions of 0.1 OD as well, due to inner filter effects?
Measuring at 0.2 enabled us to get a better signal-to-noise ratio and therefore qualitatively better data. However, this is more specific to the system we have available at our hands. In the literature, vastly different amounts are used to determine these values (e.g. OD 0.05: doi.org/10.1002/cptc.202000283; OD 0.1: 10.1002/adfm.201804227; OD 0.2:10.1002/chem.201402796).
(5) Figure S10 - The IR spectrum of RuN3 is shown; typically, azide stretching bands are very strong in intensity - why is it so weak here?
We believe the relatively weak N3 signal is due to the large amount of delocalized C-C bonds dominating the IR spectrum.
We have added the following sentence to the main text for clarification:
‘interestingly, it was observed that the IR spectrum is strongly dominated by the large amount of delocalized C-C signal’
Minor comments:
The chemical stability of RuN3 should be checked carefully as its application in living cells is discussed on page 11 and 12. Azides are known to be very reactive.
We thank the author for their comment. While we understand their concerns, the RuN3 was only used here as a control, as such we have not explored the chemical stability in biological conditions. Interestingly, the addition of the azide linker reduces the dark toxicity of the molecule compared to the parent Ru complex.
The access to the azide containing ruthenium complex appears to be very relevant to several fields of application of such complexes. This fact should be highlighted in the introduction appropriately. See for instance Zabarska et al. Dalton Trans., 2016,45, 2338-2351.
The following sentence has been added to the introduction along with the suggested reference:
‘Through addition of azide functionality the resulting complex (RuN3) may be suitable for use in a diverse range of applications beyond those explored in this study such as dye sensitized solar cells (DSSCs) and catalysis’
Round 2
Revised manuscript submitted on 07 Dit 2021![](https://www.rsc-cdn.org/pubs-core/2022.0.147/content/NewImages/icon-letter.png)
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31-Oct-2021
Dear Dr Hollenstein:
Manuscript ID: CB-ART-07-2021-000146.R1
TITLE: A ruthenium-oligonucleotide bioconjugated photosensitizing aptamer for cancer cell specific photodynamic therapy
Thank you for submitting your revised manuscript to RSC Chemical Biology. After considering the changes you have made, I am pleased to accept your manuscript for publication. I have copied any final comments from the reviewer(s) below.
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With best wishes,
Claudia Höbartner
Associate Editor, RSC Chemical Biology
Institute of Organic Chemistry, University of Würzburg
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Reviewer 2
The authors provided convincing evidence for all points I have raised. I do not have any further comments on this nice piece of work.
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