Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript describes the preparation and characterization of a celastrol-loaded liposomal hydrogel microneedle delivery system, evaluating its particle size, encapsulation efficiency, mechanical performance, and transdermal properties. Additionally, the authors investigated its therapeutic efficacy against psoriasis-like lesions using an imiquimod-induced mouse model. The study addresses an interesting topic and is well-organized and clearly written. However, the following points should be addressed to improve the quality and clarity of the manuscript before publication:

 

1. Liposome Composition: The authors should describe how the liposomal composition was selected. Specifically, please provide a clear justification for the use of PEGylated lipids, as their incorporation is typically relevant for intravenous administration rather than transdermal delivery.
2. Drug Loading Metrics: Please clarify the method used to separate the unencapsulated celastrol from the liposomes. Additionally, the authors must explicitly report both the encapsulation efficiency (EE%) and the drug loading capacity (LC%).
3. Release Kinetics Prediction: Please explain the predictive capability and relevance of using the release profile of phenol red-loaded PEGDA-AM from the hydrogel microneedles.
4. Mechanism of Action: The authors should clarify and discuss the specific cellular or molecular target of celastrol within the context of this therapeutic approach.

Author Response

Comments 1: Liposome Composition: The authors should describe how the liposomal composition was selected. Specifically, please provide a clear justification for the use of PEGylated lipids, as their incorporation is typically relevant for intravenous administration rather than transdermal delivery.

Response 1: Thank you very much for your guidance and support of our manuscript. We agree that PEGylated lipids are most commonly employed in liposomal formulations intended for intravenous administration, where polyethylene glycol chains provide a steric barrier and prolong systemic circulation.

During the design of the Cel-loaded liposome hydrogel microneedles, the inclusion of DSPE-PEG2000 was to enhance the colloidal stability of the Cel-liposome system and reduce aggregation and drug leakage during microneedle preparation and storage. In addition, PEGylation has been reported to improve the biocompatibility of nanocarrier systems. As demonstrated by Zhaoting Jiang et al., PEG modification significantly enhanced the dispersibility of PDA nanoparticles while reducing their cytotoxicity toward HaCaT cells. Therefore, DSPE-PEG2000 was incorporated into our formulation primarily to improve the physicochemical stability and biocompatibility of the liposomal system. The corresponding explanation has been added to the revised manuscript (Lines 71–75).

Comments 2: Drug Loading Metrics: Please clarify the method used to separate the unencapsulated celastrol from the liposomes. Additionally, the authors must explicitly report both the encapsulation efficiency (EE%) and the drug loading capacity (LC%).

Response 2: Thank you very much for your careful review of the manuscript. In the revised manuscript, we have explicitly described the method used to separate unencapsulated celastrol. Briefly, 0.5 mL of the liposomal suspension was subjected to ultrafiltration, and the filtrate containing the free drug was collected for quantitative analysis. In parallel, another 0.5 mL aliquot of the liposomal suspension was disrupted by sonication to determine the total celastrol content. The EE% was subsequently calculated based on the amounts of free and total drug, as detailed in lines 130-134. In addition, the method of obtaining LC% have been explicitly added in lines 141–145. The determined EE% and LC% of celastrol in the liposomal formulation were 93.90% and 5.17%, respectively, and these data have been added to Section 3.1.1.

Comments 3: Release Kinetics Prediction: Please explain the predictive capability and relevance of using the release profile of phenol red-loaded PEGDA-AM from the hydrogel microneedles.

Response 3: Thank you very much for your thorough review of our article. In preparing our response regarding the predictive value of phenol red as a model compound, we critically re-evaluated its suitability and recognized an important methodological limitation: phenol red, as a small hydrophilic tracer, differs substantially from celastrol in molecular weight, hydrophobicity, and release mechanism, and therefore its release profile has limited predictive capability for the actual drug release behavior from the liposome-loaded microneedle system. Upon recognizing this, we proactively replaced the phenol red release experiment with a more physiologically relevant approach—in vitro release testing using Franz diffusion cells mounted with excised mouse skin, which better mimics the barrier and release conditions encountered during transdermal delivery. The new data demonstrate that Cel-lipo-MNs achieve approximately 60% cumulative release of celastrol within 24 hours, confirming the favorable release capability of the hydrogel microneedle system. Accordingly, we have removed the original phenol red release data and replaced it with the celastrol release profile (Fig. 2H). We believe this revision provides a more accurate and clinically relevant assessment of the release kinetics of our therapeutic system.

Comments 4: Mechanism of Action: The authors should clarify and discuss the specific cellular or molecular target of celastrol within the context of this therapeutic approach.

Response 4: Thank you very much for your guidance on our paper. In the revised manuscript, we have expanded the discussion regarding the specific cellular and molecular targets of celastrol in the transdermal microneedle-based psoriasis therapy. Detailed modifications can be found in lines 498-509 of the revised manuscript.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

Manuscript ID: biomedicines-4353745

Title: Celastrol-Loaded Liposomal Hydrogel Microneedles for Safe and Effective Treatment of Psoriasis

Authors: Jiayi Li, Xiaoyao Fu, Zhonghuan Qu, Yanjun Yang

The authors have developed and investigated liposomal hydrogel microneedles for the transdermal delivery of celastrol, proposed for the treatment of psoriasis. The manuscript presents a series of experiments, including both in vitro and in vivo studies, to evaluate the efficacy of the proposed delivery system. A notable strength of the work is that the in vivo experiments were conducted using a relevant disease model, thereby confirming the therapeutic potential of the developed formulation. Overall, the topic is relevant to the scope of the journal. However, prior to publication, the authors should address several comments and make the necessary revisions to the manuscript.

 

1. The introduction to the article lacks a comparative analysis of liposomal systems versus other nanocarriers intended for transdermal drug delivery. What are the advantages of liposomes? A significant number of studies in the literature focus on loading celastrol into liposomal systems and microneedles. What is novel about this work?
2. Information on the experimental protocol for animal experiments is recommended.
3. Despite being mentioned in the "Results" section, the composition of the liposomes should be provided in Section 2.3. The total concentration of the liposomal formulation and the final volume should be indicated. The authors state that thin film hydration was carried out using a phosphate buffer but do not provide the pH value. Typically, the use of buffer solutions can decrease the zeta potential.
4. The manufacturer of the ultrasonic probe used to obtain unilamellar liposomes and the ultrasound processing parameters (power or amplitude) should be provided. Provide the scattering angle values of the device used to determine liposome size.
5. Hydrophobic drugs are typically loaded during the thin film preparation stage. In this study, the authors added Celastrol, which was dissolved in chloroform, to the flask containing the lipid film. Then, they removed the solvent using a rotary evaporator. How efficient is this loading method? The authors also mention the optimization of the Cel-lipo composition for EE% in the experimental section (lines 121-125). However, this optimization is not discussed elsewhere in the text, nor is any quantitative data on encapsulation efficiency provided. How did the authors calculate EE%? Celastrol is practically insoluble in water. How much does its solubility increase when it is encapsulated in liposomes? 6. The figures are placed in a separate section titled "Figures, Tables, and Schemes," which makes it inconvenient to read the paper. Figures should be placed after their first mention in the text. Furthermore, it would be advisable to separate combined figures and place them in the appropriate locations within the article.
6. In section 3.1.1. (Lines 239-242), the authors report that the liposome composition was previously optimized. However, it remains unclear how this component ratio was obtained. Did this group of authors perform the optimization and publish it previously, or did the authors rely on literature data from other researchers?
7. Provide PdI values for freshly prepared liposomes. Furthermore, when discussing liposome stability, the authors focus on size and polydispersity index while ignoring zeta potential. 9. The authors suggest that the release of the encapsulated drug may occur through swelling of the hydrogel matrix. However, this assertion is based on data obtained using phenol red (Fig. 2H). To what extent can the results presented in Fig. 2H be extrapolated to Celastrol? Why was phenol red chosen as a model compound besides ease of visualization? In this case, it would be appropriate to provide data on the release rate of encapsulated celastrol, as well as its membrane permeation (e.g., using Franz cells). Approximating the release curves with various mathematical models (e.g., Higuchi, Korsmeyer-Peppas) would allow for indirect confirmation of the proposed release mechanism.

Author Response

Comments 1: The introduction to the article lacks a comparative analysis of liposomal systems versus other nanocarriers intended for transdermal drug delivery. What are the advantages of liposomes? A significant number of studies in the literature focus on loading celastrol into liposomal systems and microneedles. What is novel about this work?

Response 1: Thank you very much for your support of our research. In the revised manuscript, we have now included a comparative discussion of liposomal systems versus other nanocarriers for transdermal drug delivery in the Introduction in lines 65-71. Liposomes were chosen as the carrier in this work because their phospholipid bilayer structure closely mimics the lipid composition of the stratum corneum, facilitating fusion with and diffusion into the skin. They offer excellent biocompatibility, the ability to encapsulate both hydrophobic and hydrophilic drugs, controlled release kinetics, and versatile surface modification (such as PEGylation) to improve stability and reduce non-specific interactions. Compared to polymeric nanoparticles, solid lipid nanoparticles, or transfersomes, liposomes provide a well-established, tunable platform that can be readily incorporated into dissolving microneedles while maintaining structural integrity. Regarding the novelty of this work, although celastrol has been separately loaded into liposomes and microneedles in previous studies, to the best of our knowledge, the integration of celastrol-loaded PEGylated liposomes with microneedles for psoriasis treatment has not been reported. The innovation lies in the synergistic combination: the microneedles physically overcome the stratum corneum barrier to deliver intact PEGylated liposomes into the viable skin, where they form a local drug depot. Importantly, the PEGylation is designed to stabilize the liposomes during the fabrication process of microneedle and to impart stealth properties that minimize premature clearance by activated phagocytes in the psoriatic microenvironment, thereby sustaining local anti-inflammatory action. These aspects together address key limitations of existing celastrol formulations and provide a novel transdermal therapeutic strategy.

Comments 2: Information on the experimental protocol for animal experiments is recommended.

Response 2: Thank you very much for your valuable comment. In the revised manuscript, we have expanded Section 2.6 “Evaluation of the therapeutic efficacy of Cel-lipo-MNs in an IMQ-induced psoriasis model,” by providing detailed descriptions of the experimental procedures, including model establishment, animal grouping, drug administration, and sample collection.

Comments 3: Despite being mentioned in the "Results" section, the composition of the liposomes should be provided in Section 2.3. The total concentration of the liposomal formulation and the final volume should be indicated. The authors state that thin film hydration was carried out using a phosphate buffer but do not provide the pH value. Typically, the use of buffer solutions can decrease the zeta potential.

Response 3: Thank you very much for your support and review of our manuscript. In the revised manuscript, we have now specified the full composition of the liposomes (optimal mass ratio of DOPC:cholesterol:Cel: DSPE-PEG2000 was determined to be 6:1:0.875:1.05.) in Section 2.3 lines 138-140. The total lipid concentration of the liposomal formulation was 9.68 mg/mL, and the final volume was 5 mL; both have been indicated in the revised method in line 128 and line 140. The phosphate buffer used for thin film hydration was at pH 7.4±0.2 in line 124, and this value has now been explicitly stated.

Comments 4: The manufacturer of the ultrasonic probe used to obtain unilamellar liposomes and the ultrasound processing parameters (power or amplitude) should be provided. Provide the scattering angle values of the device used to determine liposome size.

Response 4: We thank the reviewer for this careful observation. In the revised manuscript, we have now provided the manufacturer of the ultrasonic probe and the detailed processing parameters in Section 2.3 in lines 125-127. The probe sonication was performed using from Shanghai Bilang Instrument Manufacturing Company, ultrasonic power was set to 60% for 10min in an ice bath (pulse mode: on 15 s, off 15 s). Regarding the scattering angle, the liposome size was determined by dynamic light scattering using a Zetasizer Nano ZS90 (Malvern Instruments, UK) at a scattering angle of 90°. This information has been added to the revised manuscript in line 147.

Comments 5: Hydrophobic drugs are typically loaded during the thin film preparation stage. In this study, the authors added Celastrol, which was dissolved in chloroform, to the flask containing the lipid film. Then, they removed the solvent using a rotary evaporator. How efficient is this loading method? The authors also mention the optimization of the Cel-lipo composition for EE% in the experimental section (lines 121-125). However, this optimization is not discussed elsewhere in the text, nor is any quantitative data on encapsulation efficiency provided. How did the authors calculate EE%? Celastrol is practically insoluble in water. How much does its solubility increase when it is encapsulated in liposomes?

Response 5: Thank you very much for your valuable comments and suggestions. We have carefully considered each point and provide our responses below.

1) Optimization of liposome formulation and encapsulation efficiency：The optimization results of the Cel-lipo formulation have been explicitly described in the revised manuscript (Appendix A.1, Optimization of the Cel-lipo Formulation). The encapsulation efficiency (EE%) of celastrol was calculated as follows: EE% = (amount of encapsulated celastrol / total amount of celastrol added) × 100%. The amount of encapsulated celastrol was determined by ultrafiltration, as described in Lines 130–134 of the revised manuscript. Following formulation optimization, the EE% and LC% of the Cel-lipo system were determined to be 93.90% and 5.166%, respectively, demonstrating the effectiveness of the formulation strategy. These values have been added to the revised manuscript (Lines 281–282).

2) Improved aqueous solubility after liposomal encapsulation: Celastrol is practically insoluble in water, with a reported aqueous solubility of 13.25 μg/mL at 37 ℃. After encapsulation into our liposomes, the celastrol concentration achieved in the liposomal dispersion reached 0.470 mg/mL, corresponding to an approximately 35-fold increase in apparent aqueous solubility compared with free celastrol.

Comments 6: The figures are placed in a separate section titled "Figures, Tables, and Schemes," which makes it inconvenient to read the paper. Figures should be placed after their first mention in the text. Furthermore, it would be advisable to separate combined figures and place them in the appropriate locations within the article.

Response 6: Thank you very much for your valuable suggestions. The manuscript was prepared in accordance with the formatting requirements and template provided by Biomedicines. We appreciate the reviewer’s comment and will be pleased to adjust the figure placement if required by the editorial office.

Comments 7: In section 3.1.1. (Lines 239-242), the authors report that the liposome composition was previously optimized. However, it remains unclear how this component ratio was obtained. Did this group of authors perform the optimization and publish it previously, or did the authors rely on literature data from other researchers?

Response 7: Thank you very much for your valuable suggestions on our paper. The optimization of the Cel-lipo composition was conducted by our group and has not been published elsewhere. In the revised manuscript, we have now provided the full details of the optimization experiments, including the evaluated parameters and the results, in Appendix A.1 Optimization of the Cel-Lipo formulation in lines 563-573. The optimized component ratio was determined based on these data, and the relevant section in the manuscript now references this appendix.

Comments 8: Provide PdI values for freshly prepared liposomes. Furthermore, when discussing liposome stability, the authors focus on size and polydispersity index while ignoring zeta potential.

Response 8: Thank you very much for your professional advice. In the revised manuscript, we have included the polydispersity index (PdI) of freshly prepared liposomes. The average PdI was 0.2253, confirming a narrow size distribution and homogeneous vesicle population, which has been added in Section 3.1.1 in lines 285-286. The stability assessment of Cel-lipo focused primarily on particle size and polydispersity index (PdI), as the stability of PEGylated liposomes is predominantly governed by steric stabilization provided by the PEG corona rather than by electrostatic repulsion. In phosphate buffer (pH 7.4), the zeta potential of Cel-lipo was near-neutral (approximately -1.83±0.92 mV) due to charge screening.

Comments 9: The authors suggest that the release of the encapsulated drug may occur through swelling of the hydrogel matrix. However, this assertion is based on data obtained using phenol red (Fig. 2H). To what extent can the results presented in Fig. 2H be extrapolated to Celastrol? Why was phenol red chosen as a model compound besides ease of visualization? In this case, it would be appropriate to provide data on the release rate of encapsulated celastrol, as well as its membrane permeation (e.g., using Franz cells). Approximating the release curves with various mathematical models (e.g., Higuchi, Korsmeyer-Peppas) would allow for indirect confirmation of the proposed release mechanism.

Response 9: Thank you very much for your thorough review of our article. We acknowledge the limitation that the swelling-driven release mechanism was inferred solely from phenol red release studies. Phenol red, a hydrophilic tracer, was selected because of its ease of visualization and its suitability for evaluating the swelling and erosion behavior of the hydrogel microneedle matrix. However, phenol red cannot fully represent the release of celastrol, a hydrophobic drug encapsulated in PEGylated liposomes within the matrix. In the revised manuscript, we have addressed this by using Franz diffusion cells mounted with excised animal skin. The microneedle group achieved approximately 60% celastrol release within 24 hours. This revision has been specifically added in lines 350-354 of the revised manuscript. In addition, we have deleted the original phenol red release behavior graph and replaced it with the current release profile (Fig. 2H).

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have responded to most of the comments and made revisions to the text of the manuscript. However, some minor points still require attention.
1. The authors assessed the improvement in celastrol solubility when loaded into liposomes compared to its solubility in a water. However, these data are not presented in the main text of the manuscript. It is recommended that this information be included in the "Results" section.
2. The authors did not respond to the following comment: "Hydrophobic drugs are typically loaded during the thin film preparation stage. In this study, the authors added Celastrol, which was dissolved in chloroform, to the flask containing the lipid film. Then, they removed the solvent using a rotary evaporator. How efficient is this loading method?"
3. The optimization of the liposome formulations was based solely on encapsulation efficiency. However, did the ratio of components also affect the size of the particles, their polydispersity, and the stability of the final formulation?

Author Response

Comments 1: The authors assessed the improvement in celastrol solubility when loaded into liposomes compared to its solubility in a water. However, these data are not presented in the main text of the manuscript. It is recommended that this information be included in the "Results" section.

Response 1: Thank you very much for your support of our research. In the revised manuscript, we have now included the solubility improvement data in the "Results" Section 3.1.1 in lines 280-284. We agree that presenting this information in the Results section improves the clarity and accessibility of the findings.

Comments 2: The authors did not respond to the following comment: "Hydrophobic drugs are typically loaded during the thin film preparation stage. In this study, the authors added Celastrol, which was dissolved in chloroform, to the flask containing the lipid film. Then, they removed the solvent using a rotary evaporator. How efficient is this loading method?"

Response 2: We sincerely thank the reviewer for this important comment and apologize for the confusion caused by the inaccurate description of the liposome preparation procedure in the original manuscript. After carefully reviewing our experimental records, we found that the preparation method was incorrectly described. In the actual procedure, Celastrol, DOPC, cholesterol, and DSPE-PEG2000 were first co-dissolved in chloroform to form a homogeneous organic phase. The organic solvent was then removed by rotary evaporation to generate a drug-containing lipid film, followed by hydration with PBS to obtain Cel-lipo. Therefore, Celastrol was incorporated using the conventional thin-film hydration method for hydrophobic drugs rather than being added to a pre-formed lipid film. To avoid further misunderstanding, we have corrected the description of the liposome preparation procedure in the revised manuscript (Lines 116-126). We sincerely appreciate the reviewer for bringing this issue to our attention.

Comments 3: The optimization of the liposome formulations was based solely on encapsulation efficiency. However, did the ratio of components also affect the size of the particles, their polydispersity, and the stability of the final formulation?

Response 3: We would like to express our sincere gratitude for your thoughtful comment. We fully agree that the optimization of liposome formulations should ideally consider multiple critical quality attributes, including encapsulation efficiency, particle size, polydispersity index (PDI), and colloidal stability. In this study, the formulation screening of Cel-lipo was primarily conducted to identify a liposomal composition capable of achieving sufficient drug loading for subsequent microneedle fabrication and in vivo therapeutic evaluation. Therefore, encapsulation efficiency was selected as the primary optimization criterion during the preliminary formulation development stage. After the formulation was established, its physicochemical properties, including particle size, PDI, and storage stability, were further characterized and demonstrated favorable performance. Nevertheless, we acknowledge that variations in the lipid composition may influence not only drug encapsulation but also particle size distribution, homogeneity, and formulation stability. Incorporating these parameters into the optimization process would provide a more comprehensive basis for Cel-lipo formulation design. We have acknowledged this limitation in the revised manuscript (lines 476-481) and have noted that a more thorough optimization incorporating size, polydispersity, and stability as co-criteria will be pursued in future studies.

We would like to express our sincere thanks once again for your valuable suggestions and insightful guidance.

Author Response File: Author Response.docx