High-throughput preparation of radioprotective polymers via Hantzsch’s reaction for in vivo X-ray damage determination

Radioprotectors for acute injuries caused by large doses of ionizing radiation are vital to national security, public health and future development of humankind. Here, we develop a strategy to explore safe and efficient radioprotectors by combining Hantzsch’s reaction, high-throughput methods and polymer chemistry. A water-soluble polymer with low-cytotoxicity and an excellent anti-radiation capability has been achieved. In in vivo experiments, this polymer is even better than amifostine, which is the only approved radioprotector for clinical applications, in effectively protecting zebrafish embryos from fatally large doses of ionizing radiation (80 Gy X-ray). A mechanistic study also reveals that the radioprotective ability of this polymer originates from its ability to efficiently prevent DNA damage due to high doses of radiation. This is an initial attempt to explore polymer radioprotectors via a multi-component reaction. It allows exploiting functional polymers and provides the underlying insights to guide the design of radioprotective polymers.

1. Please revise the writing style of the word "ionizing" must be the same all over the manuscript. 2-The high light paragraphs in the results and discussion should be preferred to remove to the experimental section 3-More discussion and references are needed concerning with the level of cell necrosis , DNA damage , MAD , SOD and zebrafish larvae Please note Reviewer #2 has also provided a highlighted PDF attached.

Reviewer #3 (Remarks to the Author):
In this study, authors described the preparation and radioprotective effects of new polymers. They used cellular and zebrafish experiments for approving of theirs radioprotective effect. This study has major defects in technically experiments for evaluation of radioprotective effect. This manuscript is not suitable for publication. 1-Which part of polymer is able to scavenge free radicals; mechanisms are unclear? 2-Colony assay is necessary to do for evaluation of cellular radioprotective effects. 3-Amifostine is a prodrug (thiophosphate part of molecule should be hydrolyzed in vivo to active form) then amifostine is week in vitro for radioprotective effect, its free thiol derivative should be used. 4-Survival of irradiated mice (with and without treatment) should be used as standard in vivo model for demonstrating of radioprotective effect and calculation of DRF for both amifostine and polymers.
1) The PDI of the obtained polymers have great disparity (from 1.31 to 9.23), what is the probable reason?
Answer: Thanks for the professional question. The N,N-dimethylaniline moieties in P(4)(Y) might be the reason for high PDIs of P(4)(Y) (from 3.56 to 9.23). During polymerization, N,N-dimethylaniline groups possibly produce radical which might link other polymer chains leading to broad PDIs. This information and related references (Makromol. Chem. 178, 3221-3228 (1977); J. Macromol. Sci. Chem. A20, 789-805 (1983)) have been added in the revised manuscript. Thanks! 2) What are the criteria for choosing the structure of the monomers in this work? From the results, is there any structure or functional group that helps to improve the anti-radiation capability of the polymer, which is meaningful to develop promising radioprotector in a more efficient way.
Answer: Thanks for the insightful question. In this research, we also hoped to summarize some rules to select functional groups to enhance the antioxidant capability of Hantzsch ester. Thus, we used aldehydes with well-known antioxidant substituents (ferrocene, nitrobenzene, and phenol groups) and benzaldehyde derivatives containing electron-pull and electron-draw groups to prepare monomers and polymers. Phenol and ferrocene groups are efficient in improving antioxidant ability of polymers. However, the antioxidant ability of polymers is related but not equal to their anti-radiation capability. Cellular experiments are necessary to select radioprotective polymers from antioxidant polymers, and no law seems to be found by the current data. We believe a larger polymer library combined with theoretical calculation will be helpful to find some rules to guide the development of polymer anti-radiators in a more efficient way. We will try it in our future research. Some of this discussion has been added in revised 'Challenges and possible extensions'. Thanks a lot for your illuminating questions! Author's Response to Reviewer #2: 1) Please revise the writing style of the word "ionizing" must be the same all over the manuscript.
Answer: Thanks for the suggestion. The word 'ionizing' has been used in the revised manuscript as required. Thanks! 2) The high light paragraphs in the results and discussion should be preferred to remove to the experimental section.
Answer: Thanks for the suggestion. The high light paragraphs have been removed to the supplementary information as suggested. The manuscript looks more concise and better, many thanks! 3) More discussion and references are needed concerning with the level of cell necrosis, DNA damage, MAD, SOD and zebrafish larvae.