Development and Characterization of Biocompatible Cellulose—Tetraphenylethylene Hydrazone Self-Assembling Nanomicelles with Acidity-Triggered Release of Doxorubicin for Cancer Therapy

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This article is important as it introduces an innovative method for drug delivery utilizing α-cellulose self-assembling nanomicelles with aggregation-induced emission characteristics, potentially improving the therapeutic effectiveness of Doxorubicin in cancer therapy. It illustrates pH-dependent medication release, providing critical insights for the development of targeted therapies that reduce side effects and enhance drug delivery to tumor cells. However, the following concerns need to be addressed before accepting this article:

 

Abstract

The abstract needs to be clearer and emphasize the key findings instead of delving into detailed methodology. For instance, the abstract includes extensive details about the synthesis process.

 Introduction

The introduction should present a clearer logical flow, connecting the background to the study's objectives more explicitly.  Transition sentences are needed between the discussion of drug delivery systems and the introduction of AIEgens. For instance, after mentioning "conventional chemotherapy," a sentence like "To overcome these limitations, we explored novel AIEgens for enhanced drug delivery" would enhance clarity.

 Materials and Methods

 

Certain sections are deficient in detail essential to reproducibility, especially with synthesis and characterisation methods. In the synthesis of TPEHy, describe the precise quantities that were used in the reactions and the duration of each phase with greater clarity.

 Results

 

- Integrate data visualization, such as a graph illustrating pH-dependent drug release, rather of relying solely on descriptive text. Include a figure that directly connects drug release percentages with pH levels.

- Figure 2, clarify what each part illustrates. Instead of "Fluorescence calibration curve,"

 Discussion

 

 When discussing the pH-dependent release, it is important to refer to analogous studies that have demonstrated comparable results.

 

Author Response

Reviewer 1

This article is important as it introduces an innovative method for drug delivery utilizing α-cellulose self-assembling nanomicelles with aggregation-induced emission characteristics, potentially improving the therapeutic effectiveness of Doxorubicin in cancer therapy. It illustrates pH-dependent medication release, providing critical insights for the development of targeted therapies that reduce side effects and enhance drug delivery to tumor cells. However, the following concerns need to be addressed before accepting this article:

Abstract

Comment 1: The abstract needs to be clearer and emphasize the key findings instead of delving into detailed methodology. For instance, the abstract includes extensive details about the synthesis process.

Response 1: The Abstract has been modified as suggested by the Reviewer by emphasizing the main results of the study.

Introduction

Comment 2: The introduction should present a clearer logical flow, connecting the background to the study's objectives more explicitly.  Transition sentences are needed between the discussion of drug delivery systems and the introduction of AIEgens. For instance, after mentioning "conventional chemotherapy," a sentence like "To overcome these limitations, we explored novel AIEgens for enhanced drug delivery" would enhance clarity.

 

Response 2: The Introduction has been modified as suggested by the Reviewer.

 

Materials and Methods

 

Comment 3: Certain sections are deficient in detail essential to reproducibility, especially with synthesis and characterisation methods. In the synthesis of TPEHy, describe the precise quantities that were used in the reactions and the duration of each phase with greater clarity.

 

Respinse 3: The Methods section has been revised accordingly, including more details of the chemical reactions.

 

Results

 

Comment 4: Integrate data visualization, such as a graph illustrating pH-dependent drug release, rather of relying solely on descriptive text. Include a figure that directly connects drug release percentages with pH levels.

 

Response 4: The graph depicting pH dependent drug release has been modified adding error bars and placed in Figure 3.

 

Comment 5: Figure 2, clarify what each part illustrates. Instead of "Fluorescence calibration curve,"

 

Response 5: Figure 2 has been re-designed removing Figure 2A to avoid redundance, and removing Figure 2B considering that all described details are explicated in the text (sections 2.2 and 3.1).

 

 

 Discussion

 

Comment 6: When discussing the pH-dependent release, it is important to refer to analogous studies that have demonstrated comparable results.

 

Response 6: The discussion part has been expanded including also relevant comparation with other studies describing pH-triggered drug release.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Reviewing report

Manuscript entitled "Development and characterization of novel α-cellulose self-assembling nanomicelles with aggregation-induced emission properties for controlled release of doxorubicin"

My concern, you said alph cellulose. Right ?

How you knew its alpha not beta ?

Plesae remove the word alpha from all manuscript,. This suggestion is for you to avoid extra analysis prove. Just advice.

In addition, the work is not novel. Please remove the word.

1. Please capitalize th first letter for each keywords I mean : Cellulose; Doxorubicin; Drug delivery; Nanomicelles; Aggregation-induced emission.

2. Please write the whole name of DOX in the abstract line 22. Then make the abbreviation within brackets.

3. Please remove the dot before reference number 7 at line 51. Here : “tent.[7].”

4. In the introduction, Where is your novelity which you talked about in the title ?

5. Please, subscript numbers in the chemicalformula of chemicals under the subtitle “Chemicals”. For example, CH₃CN

Please, revise all.

In addition, its “α-cellulose” not “alpha-cellulose”.

6. At line 98m please write the exact quantity of “Zn powder, BP and BP-OH were suspended in 98 THF”

The same “TiCl4”

Please write the mls and gm numbers in the experimental part

Please, revise all the experimental part

 

7. At line 105, what you mean by “1H and 13C”?

8. At line 101, what you mean by 2 “yielding intermediate 2.”. 2 what ?

9. At line 112, please write the exact experimental method for DAC by details with reference please.

10. Where is your hint about fig . 1A in the manuscript?

11. Please increase the resolution and size of figure 1, Figure 2

12. Where is your reference for this method “Conjugation of the TPEHy derivative with DAC”?

The same “Preparation of CE-TPEHy-NMs”

13. At line 123, its λ_ex not ʎex.

14. Please re-write equations 1 and 2 again.

15. At line 181, why you chosepH 6.5 and 7.8 ? Where are the other PH ranges ? Give me a good reason or make please.

At lines 267-272, why you chose pH 7.4, 6.5, and 4.5 ?

 

16. At line 229, please re-write this « , a % of 95% «

17. How you calculated the DDA ? Please write the method with reference.

18. At line 270- 272, why « «. The results showed that at pH 7.4 the release is minimal (12.3% at 48 h), at pH 6.5 it reaches 54.5% at 48 h while at pH 4.5 the release is maximum (49.13% at 4 h, 82.4% at 48 h).   

 Why it ncrease n a PH and decrease in another. Please give me reasons with references, please.

19. At line 288, «delivered incapsulated in «. This is right ? Please revise the grammer

20. At line 290., « consistently with the fact that at pH 7.4 the release of DOX 290 from NMs is minimal as shown before. «

Why ?

Give me reason with reference. Not just observation

21.   At line 304, ts no project. Its work, manuscript, anything else. Please correct the word.

 22. At figure 2b, please draw fluorecence curves not colums.

23. N the discussion part,

- Where is your discussion about SEM and TEM and particles size ?

- Where is your dicussion aboyt the release study wiith reasons why sample rellease fast and other release slow ?

- Where is your discussion and reasons not observations about pH difference in release and why one pH release fast and other release slow ?

24. You need to make FTIR o prove the preparation of DA, and other samples

25. Until now I don’t understand what you made from fluorescence on figure 2b. Why its usefule ?

Why you made it ?

Please discuss more.

26. You conclsusion need to be improved by results not just 4 lines

27. Plesae revise the shape of the DAC chemical structure and see from which anhydroglucose you will break the ring. At figure 1.

GOOD LUCK

Author Response

Reviewer 2

 

Manuscript entitled "Development and characterization of novel α-cellulose self-assembling nanomicelles with aggregation-induced emission properties for controlled release of doxorubicin"

Comment group 1: My concern, you said alph cellulose. Right ?

How you knew its alpha not beta ?

Plesae remove the word alpha from all manuscript,. This suggestion is for you to avoid extra analysis prove. Just advice.

Response 1: We thank the Reviewer for addressing this point and removed the specification « alpha » referring to cellulose as suggested by the Reviewer.

 

Comment 2: In addition, the work is not novel. Please remove the word.

Response 2: The word “novel” was removed from the title, which was rewritten for better clarity. Although, we maintained it in the aims and conclusions part as this is the first description of the synthesis of nanomicelles obtained by self-aggregation of a cellulose-TPEHy polymer.

 

Comment group 3:

1. Please capitalize th first letter for each keywords I mean : Cellulose; Doxorubicin; Drug delivery; Nanomicelles; Aggregation-induced emission.
2. Please write the whole name of DOX in the abstract line 22. Then make the abbreviation within brackets.
3. Please remove the dot before reference number 7 at line 51. Here : “tent.[7].”

Response 3: The suggested corrections were performed.

 

Comment 4: In the introduction, Where is your novelity which you talked about in the title ?

Response 4: To Authors’ knowledge, there are few articles in literature that describe the use of Cellulose as the hydrophilic core of NMs (see references under this paragraph), but none of them employed this polymer to form nanocarriers functionalized with a tetraphenyl ethylene derivative (TPEHy) through an acid-labile hydrazone bond and loaded with Doxorubicin. In Authors’ opinion, the novelty resides in the composition of the polymer and its characterization in terms of controlled release of Doxorubicin in acidic environment that mimics tumor microenvironment (pH approx. 6.8)

Singam, A., et al., PEGylated ethyl cellulose micelles as a nanocarrier for drug delivery. RSC Adv, 2021. 11(49): p. 30532-30543.

Guo Y, Wang X, Shu X, Shen Z, Sun RC. Self-assembly and paclitaxel loading capacity of cellulose-graft-poly(lactide) nanomi-celles. J Agric Food Chem. 2012;60(15):3900-3908. doi:10.1021/jf3001873

Guo Y, Wang X, Shen Z, Shu X, Sun R. Preparation of cellulose-graft-poly(ɛ-caprolactone) nanomicelles by homogeneous ROP in ionic liquid. Carbohydr Polym. 2013;92(1):77-83. doi:10.1016/j.carbpol.2012.09.058

Yang Y, Guo Y, Sun R, Wang X. Self-assembly and β-carotene loading capacity of hydroxyethyl cellulose-graft-linoleic acid na-nomicelles. Carbohydr Polym. 2016;145:56-63. doi:10.1016/j.carbpol.2016.03.012

Li Y , Zhang J , Guo Y , et al. Cellulosic micelles as nanocapsules of liposoluble CdSe/ZnS quantum dots for bioimaging. J Mater Chem B. 2016;4(39):6454-6461. doi:10.1039/c6tb01534d

Liu D, Wu Q, Chen W, et al. A novel FK506 loaded nanomicelles consisting of amino-terminated poly(ethylene gly-col)-block-poly(D,L)-lactic acid and hydroxypropyl methylcellulose for ocular drug delivery. Int J Pharm. 2019;562:1-10. doi:10.1016/j.ijpharm.2019.03.022

Lu A, Petit E, Li S, Wang Y, Su F, Monge S. Novel thermo-responsive micelles prepared from amphiphilic hydroxypropyl methyl cellulose-block-JEFFAMINE copolymers. Int J Biol Macromol. 2019;135:38-45. doi:10.1016/j.ijbiomac.2019.05.087

 

Comment 5: Please, subscript numbers in the chemical formula of chemicals under the subtitle “Chemicals”. For example, CH₃CN

Please, revise all.

Response 5: We revised all chemical formulas accordingly.

 

Comment 6: In addition, its “α-cellulose” not “alpha-cellulose”.

Response 6: Authors removed the specification “alpha” as suggested before.

 

Comment group 7:

6. At line 98m please write the exact quantity of “Zn powder, BP and BP-OH were suspended in 98 THF”

The same “TiCl4”

Please write the mls and gm numbers in the experimental part

Please, revise all the experimental part

7. At line 105, what you mean by “1H and 13C”?
8. At line 101, what you mean by 2 “yielding intermediate 2.”. 2 what ?
9. At line 112, please write the exact experimental method for DAC by details with reference please.
10. Where is your hint about fig . 1A in the manuscript?
11. Please increase the resolution and size of figure 1, Figure 2
12. Where is your reference for this method “Conjugation of the TPEHy derivative with DAC”?

The same “Preparation of CE-TPEHy-NMs”

13. At line 123, its λ_exnot ʎex.
14. Please re-write equations 1 and 2 again.
15. At line 229, please re-write this « , a % of 95% «
16. How you calculated the DDA ? Please write the method with reference.
17. At figure 2b, please draw fluorecence curves not colums. 

 

Response 7: The entire Methods section and Figures have been revised according to Reviewers’ suggestions adding specific details about all chemical reactions with relevant references when appropriate. Figures 2A and 2B were removed as all details are already explicited in the text (sections 2.2 and 3.1). We did not add any reference for the conjugation of the TPEHy derivative with DAC phase, as this reaction was obtained by testing different conditions as it is now described with better clarity, because it has not been described before. Equations were re-written and removed from the Figures section to avoid redundance. The calculation of DDA was described adding more details. Also in this case applies the same principle as before, as we did not follow specific literature references. Rather, specific methods have been developed to perform these experiments.

 

Comment group 8:

15. At line 181, why you chose pH 6.5 and 7.8 ? Where are the other PH ranges ? Give me a good reason or make please.

At lines 267-272, why you chose pH 7.4, 6.5, and 4.5 ?

18. At line 270- 272, why « «. The results showed that at pH 7.4 the release is minimal (12.3% at 48 h), at pH 6.5 it reaches 54.5% at 48 h while at pH 4.5 the release is maximum (49.13% at 4 h, 82.4% at 48 h).   

 Why it ncrease n a PH and decrease in another. Please give me reasons with references, please.

20. At line 290., « consistently with the fact that at pH 7.4 the release of DOX 290 from NMs is minimal as shown before. «

Why ?

Give me reason with reference. Not just observation

 

Response 8: Tumors typically exhibit a slightly acidic microenvironment (ranging from 6.4 to 7.0) compared to normal tissues. Given this distinct pH difference between healthy and tumor tissues, targeting pH presents a promising strategy for enhancing selective drug release at tumor sites.

The described nanomicelles were designed including a pH-labile hydrazone bond. In fact, the covalent conjugation between DAC and TPEHy happens through the reaction of aldehydic and hydrazine groups, forming the hydrazone bond. To validate the pH-dependent drug release, we performed a controlled dialysis experiment choosing 3 different pH conditions with the aim of mimicking several physiological conditions such as blood and normal tissue (pH 7.4), the extracellular environment of tumor microenvironment (pH 6.5) and the endosomal environment (pH 4.5).

Relevant description has been added to the introduction, materials and methods (section 2.8) and discussion, including pertinent references. Reasons for different release rates and consequent antitumoral efficacy are now discussed in the discussion section.

 

Comment group 9:

19. At line 288, «delivered incapsulated in «. This is right ? Please revise the grammer
20. At line 304, ts no project. Its work, manuscript, anything else. Please correct the word.

 

Response 9: We thank the Reviewer for these corrections.

 

Comment group 10:

23. N the discussion part,

- Where is your discussion about SEM and TEM and particles size ?

- Where is your dicussion aboyt the release study wiith reasons why sample rellease fast and other release slow ?

- Where is your discussion and reasons not observations about pH difference in release and why one pH release fast and other release slow ?

 

Response 10: We thank this Reviewer for highlighting the need for enriching the Discussion part and revised it as suggested.

 

Comment 11:

24. You need to make FTIR o prove the preparation of DA, and other samples

Response 11: NMR validation was performed for TPE derivatives 1, 2 and 3 and spectra were included in a Supplementary Figure 1.

It was not possible to validate the chemical structure of DAC through NMR or FTIR analysis due to the solubility issues of the polymer. Tests using NMR at 80°C to increase the DAC solubility did not lead to additional information. Thus, for the determination of the percentage of dialdehyde in DAC (DDA%), a modified version of a previously described method consisting in NaOH titration of the HCl developed from the Schiff base formation during the reaction between DAC aldehyde groups and NH₂OH*HCl was used. We included relevant references and described the method under section 2.3.

To determine the % of derivative TPE conjugated to DAC, a spectrophotometric fluorescent method based on a calibration curve was developed and described with details under section 2.4.

 

Comment 12:

25. Until now I don’t understand what you made from fluorescence on figure 2b. Why its usefule ?

Why you made it ?

Please discuss more.

 

Response 12: The original version of Figure 2B represents the results of a spectrophotometric fluorescent method that was used to determine the aggregation of derivative 1 with different percentages of water, to validate its aggregation-induced-emission properties. Authors reviewed the Figures according to reviewers’ suggestions and agreed to remove this Figure, as all relevant information are described narratively in the text.

 

Comment 13:

26. You conclsusion need to be improved by results not just 4 lines 

Response 13: The conclusions section has been improved as suggested.

 

Comment 14:

27. Plesae revise the shape of the DAC chemical structure and see from which anhydroglucose you will break the ring. At figure 1.

Response 14: The Figure 1B representing the DAC chemical structure has been revised, and the overall quality of the Figures has been increased as suggested.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript describes the synthesis and characterization of fluorescent self-assembling nanomicelles (NMs) loaded with doxorubicin. Generally, the paper reads well. Specific comments and recommendations to the authors are listed below.

1) The information presented in figure 2 is recommended to be divided in several figures located to the corresponding results. Drug in vitro release profile lacks SD values.

2) The discussion section is recommended to be enriched outlining the effect of formulation variables or providing a comparative analysis with relevant studies.

Author Response

Reviewer 3

 

The manuscript describes the synthesis and characterization of fluorescent self-assembling nanomicelles (NMs) loaded with doxorubicin. Generally, the paper reads well. Specific comments and recommendations to the authors are listed below.

Comment 1:  The information presented in figure 2 is recommended to be divided in several figures located to the corresponding results. Drug in vitro release profile lacks SD values.

Response 1: Figure 2 has been re-designed removing Figure 2A to avoid redundance, and removing Figure 2B considering that all described details are explicated in the text (sections 2.2 and 3.1). Also, the graph depicting pH dependent drug release has been modified adding error bars and placed in Figure 3.

 

Comment 2: The discussion section is recommended to be enriched outlining the effect of formulation variables or providing a comparative analysis with relevant studies.

Response 2: We thank this Reviewer for highlighting the need for enriching the Discussion part and revised it completely as suggested.

 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have responded to all of the raised issues and the article is accepted for publication in CIMB journal.

Author Response

We thank the Reviewer for accepting our manuscript for publication in the CIMB journal.

Reviewer 2 Report

Comments and Suggestions for Authors

Reviewing report

Manuscript entitled " Development and characterization of biocompatible cellulose- TPEHy self-assembling nanomicelles with acidity-triggered release of doxorubicin for cancer therapy"

Thank you for your great efforts. I want to say thank you because I felt the team spirit inside the reply. Its not replied from just one author.  Felt it’s a team work. So, chapeau.

But,

There are some re-mentioned comments I didn’t find their reply. May be because the reply was for the grouped comments not separately. To be honest, I’m lost in some replies and didn’t find it.

So, Could you please mention the exact reply with line numbers inside the manuscript for each comment separately not grouped, please?

 

 

1. I’m still sure this method is not novel. You said no its novel and kept the word novel inside the manuscript. Right?

My prove for it is not novel :

- Wang, H., Wang, F., Deng, P., & Zhou, J. (2021). Synthesis and Fluorescent Thermoresponsive Properties of Tetraphenylethylene‐Labeled Methylcellulose. Macromolecular Rapid Communications, 42(3), 2000497.‏

- Wang, H., Ye, X., & Zhou, J. (2019). Self-assembly fluorescent cationic cellulose Nanocomplex via electrostatic interaction for the detection of Fe3+ ions. Nanomaterials, 9(2), 279.‏

 

2. Again, Please capitalize the first letter for each keywords I mean : Cellulose; Doxorubicin; Drug delivery; Nanomicelles; Aggregation-induced emission.

3. Line 103, here is dot not start “NH2NH2*H2O”

4. I know the synthesis of DAC was described n the mentioned reference but I’m still need you write the experiment please.

5. Please write equation 2 with additional brackets

LC% = W(total DOX) / (W(total DOX) + W(CE-TPEHy nanomicelles))

6. Please increase the size of Supplementary Figure 1.

7. Please inside the manuscript with mentioning the line number for each reply. Reply to these questions again:

At line 181, why you chose pH 6.5 and 7.8 ? Where are the other PH ranges ? Give me a good reason or make please.

At lines 267-272, why you chose pH 7.4, 6.5, and 4.5 ?

 

At line 270- 272, why « «. The results showed that at pH 7.4 the release is minimal (12.3% at 48 h), at pH 6.5 it reaches 54.5% at 48 h while at pH 4.5 the release is maximum (49.13% at 4 h, 82.4% at 48 h).  

 Why it ncrease n a PH and decrease in another. Please give me reasons with references, please.

 

At line 290., « consistently with the fact that at pH 7.4 the release of DOX 290 from NMs is minimal as shown before. «

Why ?

 

Give me reason with reference. Not just observation

 

8. Again, Where is your discussion about SEM and TEM and particles size ?

 GOOD LUCK

Author Response

Manuscript entitled " Development and characterization of biocompatible cellulose- TPEHy self-assembling nanomicelles with acidity-triggered release of doxorubicin for cancer therapy"

Comment 1: Thank you for your great efforts. I want to say thank you because I felt the team spirit inside the reply. Its not replied from just one author.  Felt it’s a team work. So, chapeau.

Response 1: The Authors thank this Reviewer for the suggestions and for appreciating the effort that was made to address the questions/requests. Please find the point-by-point response below. In the manuscript, all changes regarding the second round of revision are highlighted in BLUE.

 

Comment 2: But, There are some re-mentioned comments I didn’t find their reply. May be because the reply was for the grouped comments not separately. To be honest, I’m lost in some replies and didn’t find it. 

So, Could you please mention the exact reply with line numbers inside the manuscript for each comment separately not grouped, please?

Response 2: We apologize for the reduced clarity in responses by grouping some of the questions. All responses are now provided for each point separately. 

 

Comment 3: I’m still sure this method is not novel. You said no its novel and kept the word novel inside the manuscript. Right?

My prove for it is not novel :

- Wang, H., Wang, F., Deng, P., & Zhou, J. (2021). Synthesis and Fluorescent Thermoresponsive Properties of Tetraphenylethylene‐Labeled Methylcellulose. Macromolecular Rapid Communications, 42(3), 2000497.‏

- Wang, H., Ye, X., & Zhou, J. (2019). Self-assembly fluorescent cationic cellulose Nanocomplex via electrostatic interaction for the detection of Fe3+ ions. Nanomaterials, 9(2), 279.‏

Response 3: Authors accept this point. In this regard and as a result of the analysis of the papers provided, we have removed the word “novel” and included these papers in the discussion (lines 452-465). In fact, one of the main features that differentiate the nanomicelles based on cellulose and TPEHy described in the references, having aggregation induced emission properties, with the ones described in our manuscript is the presence of a pH-labile hydrazine bond that adds the pH-controlled release properties. Therefore, we highlighted this aspect in the discussion section (lines 486-487).

Comment 4: Again, Please capitalize the first letter for each keywords I mean : Cellulose; Doxorubicin; Drug delivery; Nanomicelles; Aggregation-induced emission.

Response 4: The suggested corrections were included.

 

Comment 5: Line 103, here is dot not start “NH2NH2*H2O”

Response 5: The suggested correction was performed in this formula and also in others, where appropriate.

 

Comment 6: I know the synthesis of DAC was described n the mentioned reference but I’m still need you write the experiment please.

Response 6: The details of the DAC synthesis experiment have been added (lines 169-179).

 

Comment 7: Please write equation 2 with additional brackets 

LC% = W(total DOX) / (W(total DOX) + W(CE-TPEHy nanomicelles))

Response 7: The formula has been fixed.

 

Comment 8: Please increase the size of Supplementary Figure 1.

Response 8: The size of the Figure has been increased as requested.

 

Comment 9: Please inside the manuscript with mentioning the line number for each reply. Reply to these questions again:

At line 181, why you chose pH 6.5 and 7.8 ? Where are the other PH ranges ? Give me a good reason or make please.

 

Response 9: Lines 260-264: The exact pH values tested were 7.4, 6.5, 4.5. Different conditions were reproduced, with aim of mimicking several physiological conditions such as blood and normal tissue (pH 7.4), the extracellular environment of tumor microenvironment (pH 6.5) and the endosomal environment (pH 4.5). These are commonly used pH values in studies evaluating the pH-dependent drug release from vectors, to differentiate the potential release of toxic drugs in healthy tissues or acidic solid tumor tissue. See also references:

16. Danhier, F., Feron, O., and Préat, V., To exploit the tumor microenvironment: Passive and active tumor targeting of nanocarriers for anti-cancer drug delivery. J Control Release, 2010. 148(2): p. 135-146.
17. Tannock, I.F., and Rotin, D., Acid pH in tumors and its potential for therapeutic exploitation. Cancer Res. 1989. 49(16): p. 4373-4384.
18. Ronca, R., and Supuran, C.T., Carbonic anhydrase IX: An atypical target for innovative therapies in cancer. Biochim Biophys Acta Rev Cancer, 2024. 1879(4): p. 189120.
19. Reshetnyak, Y.K., et al., Measuring tumor aggressiveness and targeting metastatic lesions with fluorescent pHLIP. Mol Imaging Biol, 2011. 13(6): p. 1146-1156.
20. Prabaharan, M., et al., Amphiphilic multi-arm-block copolymer conjugated with doxorubicin via pH-sensitive hydrazone bond for tumor-targeted drug delivery. Biomaterials, 2009. 30(29): p. 5757-5766.
21. Liu, M., et al., Novel multifunctional triple folic acid, biotin and CD44 targeting pH-sensitive nano-actiniaes for breast cancer combinational therapy. Drug Deliv. 2019. 26(1): p. 1002-1016.
22. Xiong, S., et al., A pH-sensitive prodrug strategy to co-deliver DOX and TOS in TPGS nanomicelles for tumor therapy. Colloids Surf B Biointerfaces, 2019. 173: p. 346-355.
23. Liang ,Y., et al., Preparation of pH Sensitive Pluronic-Docetaxel Conjugate Micelles to Balance the Stability and Controlled Re-lease Issues. Materials (Basel), 2015. 8(2): p. 379-391.

We added relevant description together with references in the introduction (lines 80-85) and methods (260-264) sections, and compared the results of similar studies that performed the same experiment in the discussion section (424-451) to justify this setting and the choice of pH values.

 

Comment 10: At lines 267-272, why you chose pH 7.4, 6.5, and 4.5 ?

Response 10: See previous answer.

 

Comment 11: At line 270- 272, why « «. The results showed that at pH 7.4 the release is minimal (12.3% at 48 h), at pH 6.5 it reaches 54.5% at 48 h while at pH 4.5 the release is maximum (49.13% at 4 h, 82.4% at 48 h).   

 Why it ncrease n a PH and decrease in another. Please give me reasons with references, please.

Response 11: The reason for different rates of drug release in diverse pH solutions is due to the presence of the hydrazone bond in the polymer, as result of the covalent conjugation between DAC and TPEHy. The hydrazone bond is susceptible to hydrolysis at acidic pH (such as the tumor interstitial liquid or lysosomial pH), allowing the intracellular uptake of the anticancer drug from the NMs. Hydrazone bonds have been used before in drug delivery systems and are particularly suitable for intratumoral delivery.

See relevant references:

16. Danhier, F., Feron, O., and Préat, V., To exploit the tumor microenvironment: Passive and active tumor targeting of nanocarriers for anti-cancer drug delivery. J Control Release, 2010. 148(2): p. 135-146.
17. Tannock, I.F., and Rotin, D., Acid pH in tumors and its potential for therapeutic exploitation. Cancer Res. 1989. 49(16): p. 4373-4384.
18. Ronca, R., and Supuran, C.T., Carbonic anhydrase IX: An atypical target for innovative therapies in cancer. Biochim Biophys Acta Rev Cancer, 2024. 1879(4): p. 189120.
19. Reshetnyak, Y.K., et al., Measuring tumor aggressiveness and targeting metastatic lesions with fluorescent pHLIP. Mol Imaging Biol, 2011. 13(6): p. 1146-1156.
20. Su, X., and Aprahamian, I., Hydrazone-based switches, metallo-assemblies and sensors. Chem Soc Rev, 2014. 43(6): p. 1963-1981.
21. Sonawane, S.J., Kalhapure, R.S., and Govender, T., Hydrazone linkages in pH responsive drug delivery systems. Eur J Pharm Sci, 2017. 99: p. 45-65.

 

Our results are consistent also with previous studies showing that at pH 7.4 the hydrazone bond is stable, while at lower pH values (6.8 or lower) there is an increasing hydrolysis rate at further decreasing pH values. We discussed our release rates also with reference to previous studies which employed the same or other strategies for pH-dependent release and found similar results. See discussion part lines 424-451 and references:

49. Prabaharan, M., et al., Amphiphilic multi-arm-block copolymer conjugated with doxorubicin via pH-sensitive hydrazone bond for tumor-targeted drug delivery. Biomaterials, 2009. 30(29): p. 5757-5766.
50. Liu, M., et al., Novel multifunctional triple folic acid, biotin and CD44 targeting pH-sensitive nano-actiniaes for breast cancer combinational therapy. Drug Deliv. 2019. 26(1): p. 1002-1016.
51. Xiong, S., et al., A pH-sensitive prodrug strategy to co-deliver DOX and TOS in TPGS nanomicelles for tumor therapy. Colloids Surf B Biointerfaces, 2019. 173: p. 346-355.
52. Liang ,Y., et al., Preparation of pH Sensitive Pluronic-Docetaxel Conjugate Micelles to Balance the Stability and Controlled Re-lease Issues. Materials (Basel), 2015. 8(2): p. 379-391.

 

Comment 12: At line 290., « consistently with the fact that at pH 7.4 the release of DOX 290 from NMs is minimal as shown before. «

Why ? Give me reason with reference. Not just observation

Response 12: We apologize for the lack of clarity. In fact, we added more release % values to the results part (lines 358-361) to better discuss the cell viability experiment results. As is now reported, at pH 7.4 the release of DOX encapsulated in NMs was minimal (6.7% at 4h, 7.9% at 24h and 12.7% at 48 h), at pH 6.5 it increased to 26.6% at 4h, 49.3% at 24h and 54.5% at 48 h, while at pH 4.5 the release was maximum (49.13% at 4 h, 79.9% at 24h and 82.5% at 48 h).

To determine the possible different effects of release values on cell lines, we cultured tumor cells in two different pH mediums: physiological 7.4 mimicking healthy tissues, and 6.5 mimicking acidic tumor environment. We did not culture cells at 4.5 pH because such values are incompatible with cell viability, representing an important bias to the interpretation of the results.

When cells were cultured in a medium having physiological pH 7.4, we expected the release rate of DOX from NMs to be 7.9% at 24h (the timing of the cell viability experiment), and 49.3% in the pH 6.5 medium. DOX exerts its toxicity to eukaryotic cells if there is an intracellular uptake, by provoking direct DNA damage and oxidative stress release. The viability results showed a complete cell survival in the groups treated with NMs at pH 7.4, while there was a significant decrease at pH 6.5, suggesting that there was a release of DOX from NMs and a cellular uptake at lower pH. We changed the formulation of phrases in order to increase the clarity of this section. Relevant references are the ones cited in comments above.

 

Comment 13: Again, Where is your discussion about SEM and TEM and particles size ?

Response 13: The discussion on particle size, which were confirmed via DLS and both SEM and TEM imaging, is in the lines 404-412 of the discussion.

Author Response File: Author Response.pdf

Round 3

Reviewer 2 Report

Comments and Suggestions for Authors

Reviewing report

Manuscript entitled " Development and characterization of biocompatible cellulose- TPEHy self-assembling nanomicelles with acidity-triggered release of doxorubicin for cancer therapy"

Thank you for your great efforts. Its accepted but small thing

Are you sure that Figure 2a is a SEM not TEM ?

Its Ok if it was a mistake. Mistakes happen.

Just correct what it is in reality ?

 

1. Please write the whole name of cellulose and (CE) abbreviation beside the whole name in the first mentioned place inside the manuscript.

2.  Line 174, please subscript 4 number here “NaIO₄”.

3. In the DAC preparation method which you added, you must adjyst the pH within a range. You didn’t write this part.

4. Line 407, please make a space between the number and the word “nm”. Here “20-50nm”. Please revise other similar cases.

5. Are you sure that Figure 2a is a SEM not TEM ?

GOOD LUCK

Author Response

Manuscript entitled " Development and characterization of biocompatible cellulose- TPEHy self-assembling nanomicelles with acidity-triggered release of doxorubicin for cancer therapy"

Thank you for your great efforts. Its accepted but small thing 

Are you sure that Figure 2a is a SEM not TEM ?

Its Ok if it was a mistake. Mistakes happen. 

Just correct what it is in reality ?

 

The Authors thank this Reviewer for the revision and the overall appreciation of our manuscript and the corrections that were made following the suggestions. Please find the point-by-point response below. In the manuscript version Rev_3, all changes are highlighted in RED.

 

 

1. Please write the whole name of cellulose and (CE) abbreviation beside the whole name in the first mentioned place inside the manuscript.

 

Response 1: the use of abbreviation for cellulose has been revised.

 

2. Line 174, please subscript 4 number here “NaIO₄”.

 

Response 2: the mistake has been fixed.

 

3. In the DAC preparation method which you added, you must adjyst the pH within a range. You didn’t write this part.

 

We apologize for this missing information. We now added that pH was maintained approximately 1 throughout the reaction.

 

4. Line 407, please make a space between the number and the word “nm”. Here “20-50nm”. Please revise other similar cases.

 

Response 4: the spaces between numbers and measured has been revised throughout the manuscript.

 

5. Are you sure that Figure 2a is a SEM not TEM ?

 

Response 5: Figure 2A was acquired using a SEM, and Figure 2B a TEM. I enclose with this report the original figure with the specifications of the instrument settings, which are as we indicated in the paper (images were acquired with a Quanta250 SEM (FEI, Hillsboro, USA), operating in secondary electron mode, using an acceleration voltage of 20.00 kV and a working distance (WD) of 9.3 mm). Should the Reviewer feel that the image might give rise to doubts in readers, we can remove it and keep only the image acquired with the TEM.

Author Response File: Author Response.pdf