Synthesis and Characterization of a Water-Soluble Nanomaterial via Deep Nitration of Light Fullerene C₆₀

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The title work Synthesis and Characterization of a Water-Soluble Nanomaterial via Deep Nitration of Light Fullerene C₆₀ adheres to a typical scientific structure and is well-structured. Synthesizing and characterising a new water-soluble polynitro-hydroxylated fullerene derivative, C₆₀(NO₂)₁₈(OH)₂, for possible biological uses, is the primary goal. With thorough explanations of the analytical findings and experimental protocols, the writing is generally easy to understand.

Some sections, particularly the Results, are dense with technical details and could benefit from clearer summaries or visual aids to highlight key findings. The introduction could better contextualize the novelty by comparing the proposed compound to existing fullerene derivatives more explicitly. Correct typographical errors and ensure consistent terminology (e.g., standardize "polynitro adduct" vs. "poly-nitroadduct").

Here a few questions for the authors.

Comment 1. On page 2 line 92 it is stated that ---'of titanium medical implants, with the goals of enhancing hardness,’ The paper hypothesizes that C₆₀(NO₂)₁₈(OH)₂ is promising for biomedical applications, such as titanium implant coatings. Have you conducted preliminary biocompatibility or antimicrobial tests to support these claims? If not, could you clarify plans for such studies or discuss how the compound’s thermal instability and alkaline hydrolysis might impact its practical use in physiological environments?

 

Comment 2. On results section page 3 line 100 it is explained that, ‘The synthesis conditions (7 days at 80°C), which is a bit harsh, and the paper does not discuss potential side reactions or degradation of the fullerene core. How did you monitor the reaction conditions (e.g., colour change or check via TLC plate)?

 

Comment 3. On page 7 line 175-177 the authors claimed that ‘A minor impurity peak, constituting approximately 3% of the total signal area, is detected at t = 2.7 minutes.’ Could you provide further characterization of this impurity (e.g., its chemical nature or potential impact on the compound’s properties)? How does this impurity affect the claimed purity (97–98%) and the reliability of the physicochemical data?

 

Comment 4. On page 23 line 562 the author stated that ‘yield was estimated at ~44% of the theoretical maximum’. The molecular formula C₆₀(NO₂)₁₈(OH)₂ is supported by elemental analysis, IR, and NMR, but thermal instability prevented mass spectrometry confirmation. Have you explored alternative mass spectrometry techniques (e.g., ESI-MS or MALDI-TOF with soft ionization) to validate the molecular formula? If not, how do you address potential uncertainties in the proposed structure and the reported yield (~44%)?

If the authors cannot address the methodological gaps (e.g., mass spectrometry), a more cautious interpretation of the molecular formula and yield may be needed to avoid overstatement.

Comment 5. The solubility studies show a significant salting-out effect in the presence of electrolytes like NaCl. Could you elaborate on how this affects the compound’s compatibility with physiological fluids (e.g., blood plasma, saline)? Additionally, how does the compound’s chemical lability in alkaline media impact its stability in physiological conditions, such as blood or cerebrospinal fluid?

 

Comment 6. On page 24 line 590 the authors mentioned that ‘In aqueous solution, C₆₀(NO₂)₁₈(OH)₂ exhibits hierarchical self-association.’ The hierarchical self-association (monomeric to microcolloidal states) is well-documented, but its implications for biomedical applications are not fully explored. How do you envision the formation of nano- and microcolloidal aggregates affecting the compound’s performance in applications like drug delivery or implant surface modification? Are there specific concentration ranges where these aggregates would be beneficial or detrimental?

Comments for author File: Comments.pdf

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors, dear editors,

 

The manuscript is well written and follows a clear, logical structure throughout. The authors have combined appropriate and up-to-date experimental methods and techniques, which are described in detail and well justified. The methodology section reflects a strong technical competence and provides sufficient information to ensure reproducibility.
The Results and Discussion section is well-written, easy to read, precise and concise. The obtained results are presented in a clear and accessible manner, accompanied by detailed interpretation and explanation, and supported by relevant literature, which together reflect the authors' deep understanding of the developments and key contributions within the field.  

In summary, this manuscript offers a meaningful contribution to the field of derivatised fullerene nanoparticles, and I believe it can be accepted in its current form for publication. 

Author Response

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Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

1. The manuscript is well written.
2. The team has established a direct, non-catalytic route to the previously unknown adduct C₆₀(NO₂)₁₈(OH)₂, introducing 18 nitro groups onto a single C₆₀ cage while retaining good water solubility.
3. The team has nicely integrated elemental analysis, IR, NMR, HPLC, TGA, electrochemistry, cryometry, and volumetric methods, validating the molecular formula, mapping redox and acid–base behaviour, quantifying self-association hierarchies, and identifing diffusion-instability zones.
4. The as-synthesized adduct showed (i) high solubility in physiological fluids (data should be included in the main or supplemetary manuscript), (ii) uniformly negative ζ-potentials that confer colloidal stability at tested concentrations, and (iii) significant antioxidant/reducing power though below that of ascorbic acid (data should be included in the main or supplementary manuscript). This collectively suggest its promise as a medical-grade nanomaterial for future biocompatibility and therapeutic studies (as authors suggests in this study).

3. Limitations of the study and questions that authors needs to address before the manuscript can be accepted for publication:
   1. Because the adduct decomposes under standard MS conditions, the current proof of the formula C₆₀(NO₂)₁₈(OH)₂ is entirely indirect (elemental analysis, IR, NMR, TGA, etc.). The exact substitution pattern on the C₆₀ cage and the possible presence of minor by-products remain uncertain. 

2. The compound hydrolyses irreversibly in mild alkaline media (pH ≥ 8) and aggregates above a few g L⁻¹ or in the presence of common electrolytes (e.g., 150 mM NaCl). Such conditions closely mimic blood and extracellular fluids, putting doubt on in-vivo shelf-life and performance.

Suggestion: The authors can try modifying the surface of the adduct with PEG, zwitterionic polymers and short peptides and repeat the stability assays to see which coating suppresses hydrolysis and salt-induced aggregation of the adduct.

3. The claim that the material is a “medical-grade nanomaterial” in the Conclusion section is not yet substantiated by cytotoxicity, immunogenicity, pharmacokinetic, or efficacy studies. Self-association and redox activity could generate unexpected biological responses.

Suggestion: The authors can perform MTT/LDH assays on one or multiple cell lines to test for cytotoxicity or perform hemolysis/complement activation tests using human or goat blood to test for hemocompatibility of the compound.

Author Response

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Author Response File: Author Response.pdf