Thermoreversible Gels Based on Chitosan Copolymers as “Intelligent” Drug Delivery System with Prolonged Action for Intramuscular Injection

Round 1

Reviewer 1 Report

These are some considerations to take into account about the manuscript submitted by the authors:

1.     Plagiarism has been detected in part of the methodology: section 2.2 final paragraph, section 2.5 complete and section 2.9 complete (attached plagiarism report)

2.       Section 3.5 lacks the wording of the last part

3.       Point 3.7. Studying the properties of gels formation in muscle tissue on chicken model in vitro is not defined in the methodological part of the study

4.       In vivo and in vitro have to be written in italics

5.       The discussion is very scarce, there are sections that do not present any bibliographical reference (3.7; 3.6; 3.4.1; 3.3.1) with which to discuss and others that use the same articles of the introduction

 

Comments for author File: Comments.pdf

Author Response

Dear reviewers and editors! The authors of the presented work sincerely thank you for the study of the article and writing a constructive review! All comments are taken into account. Below is a description of the changes in the work that we made during the revision.

Reviewer's question 1: 

These are some considerations to take into account about the manuscript submitted by the authors:

1. Plagiarism has been detected in part of the methodology: section 2.2 final paragraph, section 2.5 complete and section 2.9 complete (attached plagiarism report)

Authors' answer 1: 

The authors have rephrased the methodology where possible

 

Reviewer's question 2: 

2. Section 3.5 lacks the wording of the last part

Authors' answer 2: 

corrected on «BSA and DNA release from gel»

 

Reviewer's question 3: 

3. Point 3.7. Studying the properties of gels formation in muscle tissue on chicken model in vitrois not defined in the methodological part of the study

Authors' answer 3: 

Changes have been made to the manuscript

Gels formation in muscle tissue on chicken model.

The study of gel properties in chicken muscle tissue was carried out using chilled meat on bones (chicken legs) with skin. The study of gel properties in chicken muscle tissue was carried out using chilled meat on bones (chicken legs) with skin. The gel at a concentration of 0.25-1% was mixed with a 0.1% solution of tissue dye isosulfan blue of (Lymphotropin)  in PBS or 0.1 mg/mL with a solution of rhodamine 6G. The colored gel was injected at a temperature of 25 °C from a syringe to a depth of 0.5-1 cm under the skin of a chicken heated to 37 °C. Incubation at 37 °C was carried out for 2-24-48 hours to study gel formation and spreading of the dye.

 

Reviewer's question 4: 

4. In vivoand in vitro have to be written in italics

Authors' answer 4: 

Changes have been made to the manuscript

Reviewer's question 5: 

5. The discussion is very scarce, there are sections that do not present any bibliographical reference (3.7; 3.6; 3.4.1; 3.3.1) with which to discuss and others that use the same articles of the introduction

Authors' answer 5: 

The authors supplemented the discussion, including the importance of using the methods of FTIR and fluorescence spectroscopy to study the interaction of DNA + polycations with the formation of polyplexes, as well as a number of other methods. The technique using flow cytometry proposed by us for the characterization of polyplexes is new.

Sincerely yours,

Prof. Dr. Sci. Elena V. Kudryashova,

Chemical enzymology department

Lomonosov Moscow State University

Leninskie gory 1 Moscow Russia 119991

Reviewer 2 Report

The manuscript entitled “Thermoreversible gels based on chitosan copolymers as "intelligent" drug delivery system with prolong action for intramuscular injection” provides interesting ways to form polyplexes for gene delivery applications.

This article could be considered for publication after addressing the following points:

- The authors should discuss more intensively the interaction of rhodamine 6G with the polymer by citing a bit more references, especially since PEG units which are rather known as hydrophilic, their hydrophobic interaction with the dye should be more detailed.

 

- Could the BSA release experiment in Figure 8 be described more precisely? How the release percentage has been defined? The curve seemed to continue to grow at a lower slope than at the beginning, but still growing.

- Figure 9 is somehow confusing, the fluorescent aggregates are several micrometers large, so can these species be attributed to polyplexes? Control conditions with DNA alone, and without polyplexe should be provided at least as supporting information. How the authors explain the kinds of filaments appearing in Figure 9a ?

- Figure 10 is difficult to understand, it does not really show that the gel is formed and latter on degraded, it only shows that the dyes are diffusing to some extent, but the gel on itself is not visible on this picture. Furthermore, the authors should explain why two different dyes were employed, and what information can we get from that. Lastly, the use of chicken legs as a model for delivery should be discussed more deeply, and relying on literature.

- L 62: “Copolymers of polyethylene glycol (PEG), poly (lactic acid), chitosan are biodegradable and injectable nucleic acid delivery systems.” This sentence not clear

- l 150 a “e” is missing to thermoreversible

- In the title, prolong should be replaced by "prolonged"

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Reviewer 3 Report

Overall a good and interesting read. I have a few queries.

1. how about dose calculation of the formulation?

2. there should have been a comparison with a marketed product for better understanding.

3. were the chicken used in the study alive than sacrificed or how did the researchers carried out this particular experiment and how will it predict the fate of the formulation in vivo?

4. what about the toxicity of the formulation if it stays for around 30 days (as claimed in the article) in a specific tissue site?

 

English language is good but can be improved. Some typo mistakes throughout the article. 

Author Response

Dear reviewers and editors! The authors of the presented work sincerely thank you for the study of the article and writing a constructive review! All comments are taken into account. Below is a description of the changes in the work that we made during the revision.

Reviewer's question 1: 

1. how about dose calculation of the formulation?

Authors' answer 1: 

Intramuscular injection of 1 ml of 2% liquid sol, solidifying at 37C in the tissue, is assumed. A hematoma is formed, from which DNA (1 mg) is released within 20-30 days. During this time, the gel is completely destroyed.

 

Reviewer's question 2: 

2. there should have been a comparison with a marketed product for better understanding.

Authors' answer 2: 

Commercial products based on thermogels are unknown to the authors. At the moment, the direction of thermogels is actively developing and in the future, gene therapeutics based on chitosan-PEG releasing DNA can enter the market

Reviewer's question 3:

3. were the chicken used in the study alive than sacrificed or how did the researchers carried out this particular experiment and how will it predict the fate of the formulation in vivo?

Authors' answer 3: 

Changes have been made to the manuscript

Gels formation in muscle tissue on chicken model.

The study of gel properties in chicken muscle tissue was carried out using chilled meat on bones (chicken legs) with skin. The gel at a concentration of 0.25-1% was mixed with a 0.1% solution of tissue dye isosulfan blue of (Lymphotropin) in PBS or 0.1 mg/mL with a solution of rhodamine 6G. The colored gel was injected at a temperature of 25 °C from a syringe to a depth of 0.5-1 cm under the skin of a chicken heated to 37 °C. Incubation at 37 °C was carried out for 2-24-48 hours to study gel formation and spreading of the dye.

Reviewer's question 4: 

4. what about the toxicity of the formulation if it stays for around 30 days (as claimed in the article) in a specific tissue site?

Authors' answer 4: 

The studied formulations are non-toxic and biocompatible. This is known from a variety of literature data [1–6], including FDA approval of chitosan. We have previously studied the effect of copolymers on erythrocyte hemolysis – it turned out that polymers practically do not destroy erythrocytes and do not cause thrombosis. In 30 days, the gel resolves completely, and since the polymers are biocompatible, no harm is done to the body

 

Ref

1. Bhattarai, N.; Gunn, J.; Zhang, M. Chitosan-Based Hydrogels for Controlled, Localized Drug Delivery. Adv. Drug Deliv. Rev. 2010, 62, 83–99, doi:10.1016/j.addr.2009.07.019.
2. Tao, F.; Ma, S.; Tao, H.; Jin, L.; Luo, Y.; Zheng, J.; Xiang, W.; Deng, H. Chitosan-Based Drug Delivery Systems: From Synthesis Strategy to Osteomyelitis Treatment – A Review. Carbohydr. Polym. 2021, 251, 117063, doi:10.1016/j.carbpol.2020.117063.
3. Jiang, H.L.; Kim, Y.K.; Arote, R.; Nah, J.W.; Cho, M.H.; Choi, Y.J.; Akaike, T.; Cho, C.S. Chitosan-Graft-Polyethylenimine as a Gene Carrier. J. Control. Release 2007, 117, 273–280, doi:10.1016/j.jconrel.2006.10.025.
4. Pereira, P.; Pedrosa, S.S.; Correia, A.; Lima, C.F.; Olmedo, M.P.; González-Fernández, Á.; Vilanova, M.; Gama, F.M. Biocompatibility of a Self-Assembled Glycol Chitosan Nanogel. Toxicol. Vitr. 2015, 29, 638–646, doi:10.1016/j.tiv.2014.11.004.
5. Bhattarai, N.; Ramay, H.R.; Gunn, J.; Matsen, F.A.; Zhang, M. PEG-Grafted Chitosan as an Injectable Thermosensitive Hydrogel for Sustained Protein Release. J. Control. Release 2005, 103, 609–624, doi:10.1016/j.jconrel.2004.12.019.
6. Jiang, H.L.; Kim, Y.K.; Arote, R.; Jere, D.; Quan, J.S.; Yu, J.H.; Choi, Y.J.; Nah, J.W.; Cho, M.H.; Cho, C.S. Mannosylated Chitosan-Graft-Polyethylenimine as a Gene Carrier for Raw 264.7 Cell Targeting. Int. J. Pharm. 2009, 375, 133–139, doi:10.1016/j.ijpharm.2009.03.033.

 

Sincerely yours,

Prof. Dr. Sci. Elena V. Kudryashova,

Chemical enzymology department

Lomonosov Moscow State University

Leninskie gory 1 Moscow Russia 119991

E-mail: helena_koudriachova@hotmail.com

Round 2

Reviewer 2 Report

The authors have answered all the criticial points to me, so I think this paper should be accepted for publication.

Author Response

Dear Reviewer, the authors of the presented work sincerely thank you for the study of the article and writing a constructive review! All comments are taken into account.

Sincerely yours,

Prof. Dr. Sci. Elena V. Kudryashova,

Lomonosov Moscow State University

Leninskie gory 1 Moscow Russia 119991

E-mail: helena_koudriachova@hotmail.com