New N-Methylimidazole-Functionalized Chitosan Derivatives: Hemocompatibility and Antibacterial Properties
Round 1
Reviewer 1 Report
1. Minor English polishing is required.
2. Abstracts should be revised and amended with the essential information about the synthesis method.
3. The literature review should be supported with updated references about the modification of chitosan with heterocycles and their biological applications, the following references are recommended for use.
a. Synthesis, characterization, and biological evaluation of new chitosan derivative bearing diphenyl pyrazole moiety. Int. J. Biol. Macromol. 125180, (2023)
b. Synthesis and characterization of new functionalized chitosan and its antimicrobial and in-vitro release behavior from topical gel. Int. J. Biol. Macromol. 207, 242–253 (2022).
c. Development of a chitosan derivative bearing the thiadiazole moiety and evaluation of its antifungal and larvicidal efficacy. Polym. Bull. (2023) doi:10.1007/s00289-023-04765-x.
d. New thiadiazole modified chitosan derivative to control the growth of human pathogenic microbes and cancer cell lines. Sci. Rep. 1–15 (2022) doi:10.1038/s41598-022-25772-4.
4. Ln 101, “In the 0.1 M NH4” revise
5. Ln 133, what does HT-TMCs refer to?
6. Abbreviations should be defined at first mention and used consistently thereafter. For example, the first mention of “activated partial thromboplastin time” in Ln 165 but it abbreviation was mentioned in Ln 173. Revise for all.
7. Ln 174, “Stuart R., Michel A. [31]” wrong way of citation. The correct is “Stuart and Michel [31]”.
8. Ln 181, “2APTT”, what does 2 stand for?
9. Ln 188, the citation way is wrong. “Born G. [33]” should be corrected to “Born [33]”. The same mistake was found in Ln 199.
10. Ln 225, provide the full name “CuAAC”
11. Ln 226, “as we previously described in [6]” Ref. 6 is not for authors of this work. Cite the correct one.
12. Ln 238, spectrum (singular) NOT spectra (plural)
13. It would be better to write (H12, 8.79 ppm), please do for all.
14. Authors should display the IR of NMIC compared to chitosan.
15. Revise Fig. 1
16. It would be better if you provide the equations used for calculating DS.
17. Table 1 is confusing. Authors should clarify the synthesis conditions for each derivative in the text or the table.
18. Results of solubility should be discussed.
19. Ln 280, “At concentrations of 0.083 µg/mL and 0.830 µg/mL, all NMIC derivatives and NMIC2, NMIC3, NMIC4, NMIC5 at concentrations of 8.300 µg/mL did not affect blood coagulation in the BRT test (Figure 4)” by looking at Fig. 4, NMIC showed different effect.
20. Ln 287, “Addition of the studied samples to blood at concentrations of 80,000 µg/mL and 830.000 µg/mL” but in Fig. 4 83 and 830. Correct.
21. Ln 290, (187.19±10.76 c) what do c letters stand for.
22. Ln 294, “The blood clotting time increased with an increase in the degree of substitution” but from Fig. 4, NMIC did not obey this conclusion. Please, clarify and describe in detail why you found these results and compared them with previous works.
23. Generally, all found results should be discussed in detail.
24. Figs. 5-7 should be improved.
25. Ln 343, revise the range “0.174±0.170 – 1.324±0.380%”
26. Ln 344, (26 µg/mL and 250 344 µg/mL), I see it in figure8 25 µg/mL.
27. Provide the Y-title in Fig. 8
28. Conclusion should be rewritten; it is too long and some statements should be transferred to the introduction.
Minor English polishing is required.
Author Response
Dear Reviewer,
We are very grateful to you for reading and commenting on our article. Below are our responses to each of your comments.
- Minor English polishing is required.
Corrected.
- Abstracts should be revised and amended with the essential information about the synthesis method.
Corrected.
- The literature review should be supported with updated references about the modification of chitosan with heterocycles and their biological applications, the following references are recommended for use.
- Synthesis, characterization, and biological evaluation of new chitosan derivative bearing diphenyl pyrazole moiety. Int. J. Biol. Macromol. 125180, (2023)
- Synthesis and characterization of new functionalized chitosan and its antimicrobial and in-vitro release behavior from topical gel. Int. J. Biol. Macromol. 207, 242–253 (2022).
- Development of a chitosan derivative bearing the thiadiazole moiety and evaluation of its antifungal and larvicidal efficacy. Polym. Bull. (2023) doi:10.1007/s00289-023-04765-x.
- New thiadiazole modified chitosan derivative to control the growth of human pathogenic microbes and cancer cell lines. Sci. Rep. 1–15 (2022) doi:10.1038/s41598-022-25772-4.
Corrected, added new references.
- Ln 101, “In the 0.1 M NH4” revised
Corrected.
- Ln 133, what does HT-TMCs refer to?
Corrected. Changed it to NMIC.
- Abbreviations should be defined at first mention and used consistently thereafter. For example, the first mention of “activated partial thromboplastin time” in Ln 165 but it abbreviation was mentioned in Ln 173. Revise for all.
The abbreviations were corrected.
- Ln 174, “Stuart R., Michel A. [31]” wrong way of citation. The correct is “Stuart and Michel [31]”.
Corrected
- Ln 181, “2APTT”, what does 2 stand for?
In the designation of this concentration there is the number 2 and the abbreviation APTT. The number 2 means that the concentration of the sample is determined, at which the plasma coagulation time increases by 2 times, in comparison with the control (when adding a buffer instead of a sample).
- Ln 188, the citation way is wrong. “Born G. [33]” should be corrected to “Born [33]”. The same mistake was found in Ln 199.
Corrected.
- Ln 225, provide the full name “CuAAC”
Corrected. Provided it in abstract. Copper-catalyzed azide-alkyne cycloaddition (CuAAC).
- Ln 226, “as we previously described in [6]” Ref. 6 is not for authors of this work. Cite the correct one.
Corrected.
- Ln 238, spectrum (singular) NOT spectra (plural)
Corrected.
- It would be better to write (H12, 8.79 ppm), please do for all.
Corrected in text.
- Authors should display the IR of NMIC compared to chitosan.
IR spectra were not presented, but it was mentioned in text, that the main information from it − proving the conversion of azido groups at 2100 cm-1 region on our polymer. Comparison of NMIC to starting chitosan is not very informative, so we added azido-chitosan spectra in the middle.
Changes can be clearly seen on NMR spectra. Corrected and added FTIR.
- Revise Fig. 1
Fig.1. caption corrected.
- It would be better if you provide the equations used for calculating DS.
Thank you for your comment, we provided equation for calculations in text:
Where H1/H1’/H1’’ were anomeric proton of substituted, unsubstituted and acetylated units.
- Table 1 is confusing. Authors should clarify the synthesis conditions for each derivative in the text or the table.
We corrected the table. Similar derivatives were previously obtained. Synthesis and conditions are presented in materials and methods part with original reference.
- Results of solubility should be discussed.
Corrected. Introduction of positively charged hydrophilic moieties increased water solubility of the polymer in comparison to the starting chitosan and azido-derivative. Reaching DS of 18% is enough for complete solubility in wide pH at concentration 2 mg/ml.
- Ln 280, “At concentrations of 0.083 µg/mL and 0.830 µg/mL, all NMIC derivatives and NMIC2, NMIC3, NMIC4, NMIC5 at concentrations of 8.300 µg/mL did not affect blood coagulation in the BRT test (Figure 4)” by looking at Fig. 4, NMIC showed different effect.
We have corrected the sentence: At concentrations of 0.083 µg/mL and 0.830 µg/mL, all NMIC derivatives and at concentration of 8.300 µg/mL NMIC2, NMIC3, NMIC4, NMIC5 (but not NMIC1) there were no significant effect on the coagulation time in the BRT test (Figure 4).
- Ln 287, “Addition of the studied samples to blood at concentrations of 80,000 µg/mL and 830.000 µg/mL” but in Fig. 4 83 and 830. Correct.
Corrected.
- Ln 290, (187.19±10.76 c) what do c letters stand for.
Corrected to 187.19±10.76 s.
- Ln 294, “The blood clotting time increased with an increase in the degree of substitution” but from Fig. 4, NMIC did not obey this conclusion. Please, clarify and describe in detail why you found these results and compared them with previous works.
When NMIC samples were added to the blood at concentrations of 83 mcg/ml and 830 mcg/ml, a moderate positive relationship was observed between the degree of substitution and blood clotting time; correlation coefficients (r) were 0.7304 and 0.7791 (n=30; p=0), respectively. This means that with an increase in the degree of substitution of NMIC samples, the blood clotting time increased. Unfortunately, we have not found any publications on the association of anticoagulant activity of chitosan derivatives with the degree of substitution by N-methylimidazole. But in the review Dimassi et al [Dimassi, S., Tabary, N., Chai, F., Blanchemain, N., & Martel, B. (2018). Sulfonated and sulfated chitosan derivatives for biomedical applications: A review. Carbohydrate polymers, 202, 382-396.] the authors analyzed data, including the effect of sulfation on the anticoagulant activity of chitosan derivatives. In the article Yang et al [Yang J, Luo K, Li D, Yu S, Cai J, Chen L, Du Y. Preparation, characterization and in vitro anticoagulant activity of highly sulfated chitosan. Int J Biol Macromol. 2013 Jan;52:25-31. doi: 10.1016/j.ijbiomac.2012.09.027.] the authors report that highly sulfated chitosans (degree of substitution > 2.1) significantly increase the coagulation time in the APTT test. Vongchan et al [Vongchan P, Sajomsang W, Subyen D, Kongtawelert P. Anticoagulant activity of a sulfated chitosan. Carbohydr Res. 2002 Jul 16;337(13):1239-42. doi: 10.1016/s0008-6215(02)00098-8.] showed that chitosan with a degree of sulfation of 2.13 had high anticoagulant activity.
For an example, data below and the correlation coefficient between DS and blood coagulation in the BRT test (830 µg / ml):
Correlation coefficient 0,7791; p=0 (n=30)
|
DS, % |
Coagulation time BRT, s |
|
0,25 |
330,45 |
|
0,25 |
322,3 |
|
0,25 |
692,15 |
|
0,25 |
706,45 |
|
0,25 |
535,45 |
|
0,25 |
593,1 |
|
0,5 |
439,85 |
|
0,5 |
408,3 |
|
0,5 |
420 |
|
0,5 |
408,35 |
|
0,5 |
403,55 |
|
0,5 |
420,65 |
|
1 |
529,65 |
|
1 |
557,1 |
|
1 |
590,2 |
|
1 |
614,3 |
|
1 |
530,3 |
|
1 |
509,4 |
|
2 |
832,95 |
|
2 |
755,95 |
|
2 |
861,25 |
|
2 |
920,75 |
|
2 |
550,15 |
|
2 |
986,6 |
|
3 |
746,8 |
|
3 |
737,35 |
|
3 |
836,3 |
|
3 |
890,8 |
|
3 |
825,9 |
|
3 |
915,65 |
- Generally, all found results should be discussed in detail.
Corrected.
- Figs. 5-7 should be improved.
Corrected.
- Ln 343, revise the range “0.174±0.170 – 1.324±0.380%”
Corrected.
- Ln 344, (26 µg/mL and 250 344 µg/mL), I see it in figure8 25 µg/mL.
Corrected.
- Provide the Y-title in Fig. 8
Corrected.
- Conclusion should be rewritten; it is too long and some statements should be transferred to the introduction.
Corrected.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
This manuscript describes the synthesis and characterization of imidazole derivatives from low molecular weight chitosan. The degree of substitution, the solubility, the antimicrobial activity, and the effect of chitosan derivatives on blood and its components such as plasma were well studied.
This manuscript can be recommended for publication after some issues are addressed.
1. Why did the authors use a Low Molecular Weight (LMW) chitosan to synthetize NMIC derivatives? How the chitosan molecular weight relate with the imidazole chitosan derivatives synthesis/properties of NMIC?
2. Is it possible to investigate the effect of chitosan derivatives an animal model?
3. Line 72: “… natural biopolymers” is a redundancy! Moreover, chitosan is not a biopolymer but a modified polymer from a chitin biopolymer!
4. Why did not use FTIR spectroscopy to prove the successful synthesis of chitosan derivatives in this study?
5. For the Antibacterial Activity evaluation of NMIC Derivatives how many times, the tests were repeated? Note, to check the reproducibility of the results the tests should be repeated at least three times!
Author Response
Dear Reviewer,
We are very grateful to you for reading and commenting on our article. Below are our responses to each of your comments.
- Why did the authors use a Low Molecular Weight (LMW) chitosan to synthetize NMIC derivatives?How the chitosan molecular weight relate with the imidazole chitosan derivatives synthesis/properties of NMIC?
Activity of chitosan/derivatives and its molecular weight is the main topic of many studies. We choose low molecular weight chitosan by several reasons:
- High-medium molecular weight chitosan is often used in hemostatic agents. [Hu, Z.; Lu, S.; Cheng, Y.; Kong, S.; Li, S.; Li, C.; Yang, L. Investigation of the Effects of Molecular Parameters on the Hemostatic Properties of Chitosan. Molecules 2018, 23, 1–14, doi:10.3390/molecules23123147]. Our goal was to reduce blood clotting potential.
- Low molecular weight chitosan/its derivatives have much lower viscosity of solutions. The synthesis conditions are more homogenous.
III. The synthesis was carried out with ultrasonication. We have previously showed, that low molecular weight chitosan preserves its molecular weight properties in such conditions. So CuAAC under ultrasound leads to much less degradation of the polymer chain and thus preservation of the original characteristics of the macromolecules. [Lunkov, A.; Shagdarova, B.; Lyalina, T.; Dubinnyi, M.A.; Karpova, N.; Lopatin, S.; Il’ina, A.; Varlamov, V. Simple Method for Ultrasound Assisted «click» Modification of Azido-Chitosan Derivatives by CuAAC. Carbohydr. Polym. 2022, 282, 119109, doi:10.1016/j.carbpol.2022.119109]
- Is it possible to investigate the effect of chitosan derivatives an animal model?
At the next stage of our work, we plan to investigate the effect intravenous administration of chitosan derivatives to guinea pigs
- Line 72: “… natural biopolymers” is a redundancy! Moreover, chitosan is not a biopolymer but a modified polymer from a chitin biopolymer!
The definition of modern Biopolymers term is not limited to natural origin DNA, RNA, cellulose e.t.c. Many review articles consider biopolymers as polymers of natural origin/synthetic biodegradable polymers. Corrected it.
- Why did not use FTIR spectroscopy to prove the successful synthesis of chitosan derivatives in this study?
FTIR spectra were not presented, but it was mentioned in text, that the main information from it − proving the conversion of azido groups at 2100 cm-1 region on our polymer! Comparison only NMIC to starting chitosan is not very informative, so we added azido-chitosan spectra in the middle. Changes can be clearly seen on NMR spectra − superior analytical method (especially, since all polymers are soluble in common solvent for NMR!). Corrected and added IR for additional information.
- For the Antibacterial Activity evaluation of NMIC Derivatives how many times, the tests were repeated? Note, to check the reproducibility of the results the tests should be repeated at least three times!
Added to the text: each assessment was performed at least three times to ensure reproducibility of results.
Round 2
Reviewer 1 Report
The authors addressed all issues, and now the revised version meets the publication standards.
