Review Reports

Response to Reviewer 1 Comments

1. Summary

Thank you very much for taking the time to review this manuscript. We have read each of your valuable comments carefully and revised our manuscript according to your suggestions. Please find the detailed responses below. In the resubmitted file, we have made the corresponding revisions in highlighted form. 

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Can be improved

This part has been improved and added to the manuscript. Thank you very much for your comments.

Are all the cited references relevant to the research?

Yes

Thank you very much for your approval in this respect.

Is the research design appropriate?

Must be improved

With regard to this section, we have appropriately added the relevant missing methods to the manuscript and marked it in red. Thank you for your comments.

Are the methods adequately described?

Can be improved

Thank you for your suggestions in this regard. We have made corresponding improvements.

Are the results clearly presented?

Yes

Thank you very much for your affirmation of our part.

Are the conclusions supported by the results?

Must be improved

With regard to this part, we have made corresponding improvements. Thank you very much for your suggestions.

3. Point-by-point response to Comments and Suggestions for Authors

· Comments 1: The abstract requires improvement regarding cohesion, appropriate use of abbreviations and, most importantly, alignment between the reported results and the methodologies described.

Response 1: Thank you for pointing this out. We agree with this comment. We have revised the abstract section and have highlighted it in bright red in the manuscript.We have added changes on lines 13-27 of the abstract on the first page of the manuscript.Thank you again.

· Comments 2: The entire manuscript requires English language revision, since was observed repeated words (e.g., line 55), improper use of abbreviations, and spelling errors.

Response 2: Thank you for pointing out this point, and we agree with this comment, so correct it in 57, 58 and 62 in the introduction on page 2, and fill in the acronym 1227 on page 33, except for the first use of the full name will appear in the form of acronym, marked in red, please refer to the manuscript, thank you.

· Comments 3: The introduction could be strengthened by including global epidemiological data about rheumatoid arthritis.

Response 3: Thank you for pointing out this point, and we agree with this comment, so the corresponding global epidemiological data of rheumatoid arthritis have been added to lines 34-37 on the first page of the manuscript, marked in red, please refer to the manuscript, thank you.

· Comments 4: The information presented in lines 67–80 is unclear and should be revised to ensure consistency with the preceding content of the introduction. Additionally, no information regarding adalimumab is provided in the introduction.

Response 4: Thank you for pointing out this point, and we agree with this comment, so we have revised this section and added information about adamumab (ADA) to be corrected in line 80-97 of the introduction on page 2-3, details have been added to the manuscript and marked in red, thank you again.

· Comments 5: The results are generally well presented; however, some results are not described in the methodology section, such as the release study of Rhodamine B, which represents a serious flaw.

Response 5: Thank you for pointing this out, and we agree with this comment, so on page 26, 4.2.5. Line 893-901 under this item has added relevant information and marked it in red. Please refer to the manuscript. Thank you.

· Comments 6: All figures require improved quality.

Response 6: The clarity of the figures in the manuscript is limited. We have uploaded the figures separately for your review. Thank you.

· Comments 7: The tables and their footnotes should be revised to clarify the abbreviations used.

Response 7: According to your requirements, we have added the table annotations. Please refer to the manuscript for details. We have added changes on lines 184 of the table 1 on the page 5 of the manuscript. Thank you for your valuable suggestions.

· Comments 8: Table 7 and Figure 8 present the same results, leading to unnecessary redundancy.

Response 8: Thank you for pointing this out, and we agree with this comment, so we have deleted the data in Table 7. Thank you again.

· Comments 9: The discussion mainly reiterates results already presented and information from the introduction. Therefore, this section should be substantially improved by emphasizing the relevance of the findings, providing comparisons with the literature, and highlighting the scientific significance of the results.

Response 9: Following your suggestion, we have reorganized the discussion content and highlighted it in red. We have added changes on lines 593-726 of the discussion on the page 19-22 of the manuscript.Thank you.

· Comments 10: Why was adalimumab (ADA) not used in the in vitro studies?

Response 10: Thank you for pointing out this point of view, and we agree with this comment, because the in vitro study is mainly around the prepared new dosage form, that is, Dual-network hydrogel microneedle patch loaded with methotrexate nanocrystals （DHMN@MTX-NCs）, so adamumab （ADA） was not added in the in vitro experiment, but subsequent cell experiments and animal experiments were added to carry out the study of the combination of DHMN@MTX-NCs and ADA. On the surface, the effect of the combination of the two will be better than that of using them alone. To provide a new treatment for rheumatoid arthritis. Thank you again for this comment.

· Comments 11: Please include detailed information regarding the centrifugation, lyophilization, and dialysis procedures performed in Section 4.2.1, as well as insert Equation 1.

Response 11: Thank you for pointing out this point of view, and we agree with this comment. With regard to formula (1), we changed it from the original conclusion to the experimental method and inserted it into lines 788-790 on page 23.

· Comments 12: Section 4.2.3 lacks details regarding the DSC parameters and the HPLC method used. Was the method validated?

Response 12: Thank you for your comments. DSC is not used to check the entrapment efficiency of the dosage form, but to determine whether the nanocrystals have been synthesized successfully. High performance liquid chromatographic conditions have been added to page 24 lines 828-832 under item 4.2.3, and detailed DSC steps for page 24 lines 833-838 in item 4.2.3.1 have been added. The method in the DSC scan test of 4.2.4 is the same as under item 4.2.3.1, which has been added to line 856 red in the manuscript 4.2.4. Please check it. Thank you for your comments.

· Comments 13: The methodology related to mechanical properties, stability, and swelling behavior is missing.

Response 13: Thank you for your comments, and we agree with you. The experimental method has been added to item 4.2.4.1.-4.2.4.3. on pages 25 and 26, from lines 859 to 892, marked red. Thank you again for your comments.

· Comments 14: In Section 4.2.6, was human skin used? Did the authors quantify the amount of methotrexate (MTX) retained in the skin?

Response 14: In the manuscript, the skin used in the experimental part of 4.2.6 is rat skin, not human skin. We sincerely appreciate the valuable comments from the reviewers. We fully agree that quantifying the drug retention in the skin is crucial for a comprehensive assessment of the local targeting ability of the delivery system. The in vitro transdermal experiments in the early stage of this study mainly focused on verifying the basic delivery capacity of microneedles to overcome the skin barrier, and thus only the cumulative permeation amount was measured. We acknowledge that this is a limitation of the current work. However, the existing in vivo pharmacodynamic data (superior efficacy compared to traditional creams) and the sustained-release characteristics of the material both indirectly support the effective accumulation of the drug in the local area. We will follow your suggestion and directly measure the drug retention in the skin tissue in the subsequent in vivo pharmacokinetic study to strengthen the evidence chain.

4. Response to Comments on the Quality of English Language

Point 1: The entire manuscript requires English language revision, since was observed repeated words (e.g., line 55), improper use of abbreviations, and spelling errors.

Response 1: In terms of language repetition, jumble and errors, we will improve and mark them in red to avoid similar problems as far as possible. Thank you very much for your comments on us, which is very important to us. Thank you.

5. Additional clarifications

You are a serious and careful person, and you have patiently given us a lot of suggestions, which are very important to us. Thank you for your comments on us. And I wish you. Have a nice day.

Response to Reviewer 2 Comments

1. Summary

Thank you very much for taking the time to review this manuscript. We have read each of your valuable comments carefully and revised our manuscript according to your suggestions. Please find the detailed responses below. In the resubmitted file, we have made the corresponding revisions in highlighted form.

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Can be improved

This part has been improved and added to the manuscript. Thank you very much for your comments.

Are all the cited references relevant to the research?

Yes

Thank you very much for your approval in this respect. I wish you a happy life.

Is the research design appropriate?

Yes

I'm glad you agree with our part of the description. Have a nice life.

Are the methods adequately described?

Can be improved

Thank you for your suggestions in this regard. We have made corresponding improvements.

Are the results clearly presented?

Yes

Thank you very much for your affirmation of our part.

Are the conclusions supported by the results?

Yes

Thank you for your recognition of our results. Have a nice day.

3. Point-by-point response to Comments and Suggestions for Authors

· Comments 1: It is unclear whether ADA was administered systemically or locally, and how its dosing regimen relates to clinical practice.

Response 1: Thank you for your valuable comment. We would like to clarify this point. According to the 2024 edition of the Chinese Guidelines for the Diagnosis and Treatment of Rheumatoid Arthritis, TNF-α inhibitors are currently the most extensively used and well-established biological disease-modifying antirheumatic drugs (bDMARDs) for the treatment of RA. TNF-α inhibitors available in China include the monoclonal antibody adalimumab (ADA), which has ample evidence to support its efficacy and safety in the treatment of RA [1-5]. When TNF-α inhibitors are used for the treatment of RA, it is recommended to combine them with a traditional synthetic disease-modifying antirheumatic drug (csDMARD). Methotrexate is the preferred csDMARD for the treatment of RA, so the combination of the two is chosen for the treatment of RA. Thank you again for your comment.

1. National Clinical Research Center for Dermatologic and Immunologic Diseases (Peking Union Medical College Hospital); Chinese Association of Rheumatology and Immunology Physicians; Rheumatology and Immunology Professional Committee of Chinese Rehabilitation Medical Association; Rheumatology and Immunology Professional Committee of Chinese Research Hospital Association; Rheumatology and Immunology Branch of Beijing Association of Holistic Integrative Medicine. Zhonghua Nei Ke Za Zhi. 2024, 63, 1059-1077. doi:10.3760/cma.j.cn112138-20240531-00360

2. Weinblatt, M. E.； Keystone, E. C.； Furst, D. E.； Moreland, L. W.； Weisman, M. H.； Birbara, C. A.； Teoh, L. A.； Fischkoff, S. A.； Chartash, E. K. Adalimumab, a fully human anti-tumor necrosis factor alpha monoclonal antibody, for the treatment of rheumatoid arthritis in patients taking concomitant methotrexate: the ARMADA trial. Arthritis Rheum. 2003, 48, 35-45. doi:10.1002/art.10697

3. Breedveld, F. C.; Weisman, M. H.; Kavanaugh, A. F.; Cohen, S. B.; Pavelka, K.; van Vollenhoven, R.; Sharp, J.; Perez, J. L.; Spencer-Green, G. T. The PREMIER study: A multicenter, randomized, double-blind clinical trial of combination therapy with adalimumab plus methotrexate versus methotrexate alone or adalimumab alone in patients with early, aggressive rheumatoid arthritis who had not had previous methotrexate treatment. Arthritis Rheum. 2006, 54, 26-37. doi:10.1002/art.21519

4. Fleischmann, R.; Mysler, E.; Hall, S.; Kivitz, A. J.; Moots, R. J.; Luo, Z.; DeMasi, R.; Soma, K.; Zhang, R.; Takiya, L.; Tatulych, S.; Mojcik, C.; Krishnaswami, S.; Menon, S.; Smolen, J. S.; ORAL Strategy investigators. Efficacy and safety of tofacitinib monotherapy, tofacitinib with methotrexate, and adalimumab with methotrexate in patients with rheumatoid arthritis (ORAL Strategy): a phase 3b/4, double-blind, head-to-head, randomised controlled trial. Lancet. 2017, 390, 457-468. doi:10.1016/S0140-6736(17)31618-5

5. Combe, B.; Kivitz, A.; Tanaka, Y.; van der Heijde, D.; Simon, J. A.; Baraf, H. S. B.; Kumar, U.; Matzkies, F.; Bartok, B.; Ye, L.; Guo, Y.; Tasset, C.; Sundy, J. S.; Jahreis, A.; Genovese, M. C.; Mozaffarian, N.; Landewé, R. B. M.; Bae, S. C.; Keystone, E. C.; Nash, P. Filgotinib versus placebo or adalimumab in patients with rheumatoid arthritis and inadequate response to methotrexate: a phase III randomised clinical trial. Ann Rheum Dis. 2021, 80, 848-858. doi:10.1136/annrheumdis-2020-219214

· Comments 2: The authors should justify the chosen sample size or acknowledge this limitation.

Response 2: Thank you for your comments, and we agree with you. With regard to the selection of the number of samples, we made reference to a number of literatures, and finally selected the number of samples in each group of 6. We admit that the experimental results may have limitations. Thank you very much for your advice.

· Comments 3: The authors should expand on how MTX-NCs delivered via microneedles might differentially modulate macrophage polarization compared to free MTX.

Response 3: We are grateful to the reviewer for raising this highly insightful scientific question. We fully agree that clarifying the specific regulation of MTX nanocarriers on macrophage polarization will more profoundly reveal the unique advantages of our delivery system in improving the immune microenvironment of rheumatoid arthritis (RA). We will supplement this in the discussion section. We have added changes on lines 610-623 and line 651-661 of the discussion on the page 19-20 of the manuscript.

· Comments 4: The potential role of sustained local MTX exposure in altering inflammatory signaling pathways should be discussed in more depth.

Response 4: Thank you for your comments, and we agree with you that the reason for an in-depth discussion of the impact of signal pathways. We have added changes on lines 665-693 of the discussion on the page 21 of the manuscript.

· Comments 5: To further strengthen the biological and translational framework of the study, I recommend expanding the discussion of molecular mechanisms involved in RA pathogenesis and disease activity. In particular, recent evidence highlights the relevance of signaling pathways and intracellular processes that regulate inflammation, bone remodeling, and immune activation, which are highly pertinent to the therapeutic outcomes observed in this work. doi:10.1186/s12967-025-06174-2 and doi:10.3390/ijms241612764.

Response 5: First of all, we appreciate your comments, and we agree with you. We introduce these two articles into the discussion section and mark them for Ref. 39 and Ref. 40, details of which can be found on pages 20 and 21 of the manuscript. Thank you for your advice.