Surface Modification of Cellulose Nanocrystals (CNCs) to Form a Biocompatible, Stable, and Hydrophilic Substrate for MRI
Round 1
Reviewer 1 Report
The authors had study on Surface Modification of Cellulose Nanocrystals (CNCs) to Form a Biocompatible, Stable, and Hydrophilic Substrate for MRI.
The manuscripts needs some modifications:
The main question addressed by the research is the possibility of Cellulose Nanocrystals Modification. The authors should mention the main aim in the abstract. The abstract should be re-written. It should be include all sections for an abstract.
The introduction is so long. It also not include all relevant references. Some new and relevant references should be added. The authors should also clarify the gap of study in the last paragraph of the introduction section.
The subject is not so novel, however, Modification of Cellulose Nanocrystals is new. The previous studies focused on other materials or free Cellulose.
The study design and methods are proper. However, which statistical methods has been used for analysis? The main point is to add a statistical section clearly. Then, we can understand the groups and controls.
The references are appropriate. However, for the introduction section more recent and relevant references are needed.Please improve typo errors. for example in the caption of Figure 6.
Besides, please improve the quality of TEM images.
Author Response
The authors are grateful to the reviewers for taking the time to review our manuscript and giving us insightful and helpful feedback. These comments helped us improve and strengthen our paper before publication. Regarding the feedback, the authors have submitted the manuscript's final copy, marked the text's changes in red, and carefully addressed all the reviewers' comments in a point-by-point list clearly stating where we have made changes in the revised manuscript version.
Author Response File: 
Author Response.docx
Reviewer 2 Report
This paper dealt with the surface modification of cellulose nanocrystals, and the authors propose a new medical use. So that it could be describe that this paper is enthusiastic approach. However, the characterization of the CNCs PEG/NaOH were not clear.
Comments for author File: Comments.docx
Author Response
The authors are grateful to the reviewers for taking the time to review our manuscript and giving us insightful and helpful feedback. These comments helped us improve and strengthen our paper before publication. Regarding the feedback, the authors have submitted the manuscript's final copy, marked the text's changes in red, and carefully addressed all the reviewers' comments in a point-by-point list clearly stating where we have made changes in the revised manuscript version.
Author Response File: Author Response.docx
Reviewer 3 Report
The manuscript «Surface Modification of Cellulose Nanocrystals (CNCs) to Form a Biocompatible, Stable, and Hydrophilic Substrate for MRI» submitted by Fathyah Whba et al. is devoted to preparation, analysis and characterization of cellulose nanocrystals. Authors applied acid hydrolysis of microcrystalline cellulose by sulphuric acid to obtain cellulose Nanocrystals (CNCs). The X-ray diffraction (XRD), zeta potential analysis, the transmission electron microscopy (TEM), field emission scanning electron microscopy (FESEM), FTIR and NMR methods were used for nanoparticle characterization. Conjugates of CNCs with polyethylene glycol were synthesized and analyzed. Their biocompatibility was demonstrated. Authors tested modified CNCs as a dual contrast agent for MRI in vitro studies.
Introduction is devoted to the description of CNCs preparation methods and development of MRI agents of new generation. Methods contains detailed description of phisico-chemical methods used in the work. Results are well illustrated and discussed. Conclusions are correspond to the results of work.
This manuscript will be interesting for the specialist in contrast MRI agent creation area.
I have some questions and remarks:
1) Abstract must be improved. Abstract contains too much experimental details. I think it is necessary to change abstract.
2) What does CNCs-BEG/NaOH mean?
3) The references (line 67) are not situated in order.
4) The authors write that they use 64% sulfuric acid to obtain CNCs, but they used diluted sulfuric acid (see methods 2.2.1 line 153). After dilution by water 50 ml was added to 100ml of MCC suspension. The final concentration must be no more than 21,3%. Please comment this moment and change the text.
5) Figure 2 capture is not correspond to the image. I think images illustrate the stages of the process of the CNCs synthesis.
6) Please add the reaction between PEG and CNCs surface in the presence of NaOH. What groups of cellulose are participate in covalent linkage formation.
7) Please add the abbreviation decoding when you use the abbreviation for the first time. (FTIR, TGA/DTG).
8) Do you use any control contrast agent for MRI for in vitro MRI studies? Please add this information in the manuscript.
9) What type of cell survival test did you use? Please add this information in section 2.3.9.
10) It is necessary to give the decoding of symbols used in equation 2.
11) There are no description of equation on the line 319. Please add the information about it in the text.
12) Please decode abbreviation in table 1,2 and 3 and equation 5 and 6 (hkl, dhkl, FWHM, UDM etc.).
13) Please check the capture to the figure on the page 12 (line 374). The number is absent and the capture is not completed.
14) Figure 7, I did not find panel a and b. There are 6 panel on this figure. Please change this figure for understandable variant.
15) Figure 10. Check the capture. Authors scared the description for panel a and b.
16) Conclusions as the Abstract contains a lot of experimental details. It is necessary to rewrite the conclusions based on the main finding and prospects of this work.
Moderate editing of English language is necessary, Manuscript contains some mistakes, mistapings and unhappy expressions.
Author Response
The authors are grateful to the reviewers for taking the time to review our manuscript and giving us insightful and helpful feedback. These comments helped us improve and strengthen our paper before publication. Regarding the feedback, the authors have submitted the manuscript's final copy, marked the text's changes in red, and carefully addressed all the reviewers' comments in a point-by-point list clearly stating where we have made changes in the revised manuscript version.
Author Response File: Author Response.docx
Reviewer 4 Report
In this manuscript the authors document their work on synthesizing cellulose nanocrystals (CNCs) from commercially available microcrystalline cellulose (MCC) using an acid treatment process. The eventual goal, as stated by the authors, is to use these CNCs to produce MRI contrast agents for biomedical applications. I did not find the preparation of the CNCs to be sufficient novelty as the authors have not used any particular method which is significantly different from the existing protocols in the literature. The novelty could lie in the use of the CNCs as MRI contrast agents. However, even in this context the authors have not sufficiently demonstrated its advantages. In contrast, the authors do a good job in characterizing the CNCs and the CNC-PEG/NaOH composites and solutions.
Major issues
1. The authors use the CNC-PEG/NaOH as MRI contrast agents towards the end of the paper. However, I do not understand how they might work as MRI contrast agents as they do not mention using any paramagnetic relaxation enhancers (PREs) such as Gd3+. While reading this paper, it appeared to me that the intention was to use the CNC-PEG/NaOH solutions to impregnate PREs into the nanocrystals and thereby increase their stability and bioavailability, but I did not find any reference to PREs being used in the manuscript. The authors claim that they see an enhancement in Figure 13, but I am not convinced about this claim in Fig 13a (related to T1 contrast) and it seems like a very modest enhancement in Fig 13b (related to T2 contrast, which the authors themselves don’t seem to targeting). The increase in T2 brightness in the images might just be due to the sample being more viscous or due to the presence of salts in the solution. Even if I accept the authors’ claims about T1 enhancement and assume that they forgot to mention the PRE used, they have not quantified the enhancement, and there aren’t sufficient controls to convincingly show that the incorporation of the CNCs was beneficial.
2. The cytotoxicity studies on the Hep G2 cells are not properly described and lacks sufficient controls. How are the authors defining Relative Viability %? Relative to what conditions? How is the relative viability greater than 100% at around 12.5 ug/mL of CNC-PEG/NaOH in Fig 14? What are the error bars associated with the data? The text associated with this data is also a bit confusingly worded.
Minor issues
1. In Figure 12, the authors should zoom in on the graph to show the zeta-potential more clearly?
2. Did the authors do any MALS or DLS studies to characterize the size of the nanoparticles in solution? It seems to me that they should be able to do DLS easily on their existing Malvern Zetasizer instrument.
3. In general, the figure captions should give more details about the figure than presented here.
4. In Figure 4, why is the %Transmittance higher than 100%? There might be baseline issues with the IR as well.
5. The TEM images in Figure 6 (there is an error in the numbering) are not very clear, the authors should try to acquire better images.
Author Response
The authors are grateful to the reviewers for taking the time to review our manuscript and giving us insightful and helpful feedback. These comments helped us improve and strengthen our paper before publication. Regarding the feedback, the authors have submitted the manuscript's final copy, marked the text's changes in red, and carefully addressed all the reviewers' comments in a point-by-point list clearly stating where we have made changes in the revised manuscript version.
Author Response File: Author Response.docx
Round 2
Reviewer 1 Report
Thanks for modifications. The modifications are good. It can be accepted.
Author Response
Thank you for your positive response
Reviewer 3 Report
The manuscript «Surface Modification of Cellulose Nanocrystals (CNCs) to Form a Biocompatible, Stable, and Hydrophilic Substrate for MRI» resubmitted by Fathyah Whba et al. is devoted to preparation, analysis and characterization of cellulose nanocrystals. Authors improved the manuscript with correspondence of the remarks.
This manuscript will be interesting for the specialist in contrast MRI agent creation area.
I have some additional questions and remarks:
1) Authors use the abbreviation BEG (abstract, conclusions, Lines 16, 22,25,116 etc) and PEG for polyethylene glycol. Please check all manuscript and use the correct abbreviation (PEG)
2) Figure 3. The number of ethylene glycol monomer in the PEG structure is absent. Please add this number. Analyzing the structure of the product I think that you use simple ethylene glycol instead of PEG. May be the staples and the number of monomers were missed?
3) The reference on the equation 6 is absent in the text of manuscript
4) Figure 7 and 8. Could you please not use the same symbol for image panel designation. In this figure 7 there are two (a) panel and two (b) panel. In this figure 8 there are three (a) panel and three (b) panel.
5) Figure 15. Please write in description what is represented on the image. Are there cells or drop?
Manuscript contains some mistakes and mistypings
Author Response
The authors are grateful to the reviewers for taking the time to review our manuscript and giving us insightful and helpful feedback. These comments helped us improve and strengthen our paper before publication. Regarding the feedback, the authors have submitted the manuscript's final copy, marked the text's changes in red, and carefully addressed all the reviewers' comments in a point-by-point list clearly stating where we have made changes in the revised manuscript version
Author Response File: Author Response.docx
Reviewer 4 Report
The authors have made extensive revisions to their manuscript. They have improved their methods section, added descriptions of variables in equations, and tables, and explained their goal better in this revision. A few minor issues remain:
1. Figure 2 and 4 captions need rewriting.
2. Figure 7 has a formatting error.
3. Sometimes the authors are writing CNC-BEG/NaOH instead of CNC-PEG/NaOH.
Some grammatical or oddly phrased sentences still exist. The authors should revise the manuscript thoroughly. A few examples of odd sentences are provided below:
1. See point 1 above: The captions for Fig 2 and 4 are oddly worded.
2. Line 428: "Thus, Sulfate content is thus one of the factors affecting the thermal 428 stability of prepared CNCs." - thus repeated twice in the sentence, sulfate is capitalized.
3. Line 605: hydrolysis of what?
4. Line 198: "Using a very dilute solution was 198 made by taking..." The "Using" at the start of the sentence should be deleted.
Author Response
The authors are grateful to the reviewers for taking the time to review our manuscript and giving us insightful and helpful feedback. These comments helped us improve and strengthen our paper before publication. Regarding the feedback, the authors have submitted the manuscript's final copy, marked the text's changes in red, and carefully addressed all the reviewers' comments in a point-by-point list clearly stating where we have made changes in the revised manuscript version
Author Response File: Author Response.docx
