The Therapeutic Potential of Exosomes in Ocular Surface Diseases

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript provides a comprehensive and up-to-date overview of the therapeutic potential of exosomes in ocular surface diseases. The review is well structured, mechanistically detailed, and covers a wide spectrum of preclinical evidence across dry eye disease, corneal injury, and related conditions. While the manuscript includes analyses of human-derived samples, all therapeutic data are preclinical, and no interventional human clinical trials are presented. A clearer distinction between observational human studies and experimental therapeutic models would strengthen the manuscript.

Comments and Suggestions

1. Clarification of Evidence Level (Preclinical vs Clinical)

The manuscript includes studies involving human-derived samples, such as tear extracellular vesicle profiling in dry eye disease and keratoconus, as well as plasma exosome analyses in primary Sjögren’s syndrome and diabetes. However, these are observational or mechanistic studies, not therapeutic clinical trials.

All therapeutic evidence described in the review is based on:
In vitro cell culture systems (e.g., HCECs, HCEnCs)
Animal models (mouse, rat, rabbit, porcine, feline)
Ex vivo experimental models

There are no completed human interventional trials assessing exosome-based therapy for ocular surface diseases. This distinction should be stated explicitly and clearly in both the abstract and the conclusion. The manuscript should emphasise that current therapeutic evidence remains entirely preclinical.

A table summarising for each study: human sample analysis vs in vitro vs in vivo model, intervention, mechanism, and outcome would greatly improve clarity.

2. Need for Summary Tables

The manuscript is dense and mechanistically rich, but readability and clinical usability would be significantly improved by structured tables. Suggested tables:

Table 1: Exosome source, disease model, experimental system (human sample analysis / in vitro / in vivo), key mechanism, main outcomes.
Table 2: Engineered exosomes (miRNA-loaded, nanoparticle-modified, hydrogel systems) and their translational feasibility.
Table 3: Isolation methods – advantages, disadvantages, scalability, and suitability for clinical-grade production.
Table 4: Duration of therapeutic effect observed in animal studies, including dosing frequency.

These tables would make the review far more accessible to clinicians and translational researchers.

3. Duration of Therapeutic Effect

The manuscript does not clearly summarise how long therapeutic benefits persist in animal models.

It remains unclear:
Whether improvements are transient (days to weeks) or sustained.
Whether repeated administration is required.
Whether structural restoration translates into long-term functional recovery.

Without such information, translational feasibility is difficult to assess. A dedicated subsection summarising duration of effect, dosing regimens, and follow-up periods in animal studies is recommended.

4. Dedicated Section: Translation to Clinical Practice

A separate section titled, for example, “Translational Considerations and Clinical Implementation” is needed.

This section should address:
Regulatory challenges and GMP production requirements
Batch-to-batch consistency and standardisation
Compliance with MISEV2023 guidelines
Storage stability and formulation (eye drops, hydrogels, subconjunctival injections)
Optimal dosing strategies
Safety concerns, including immunogenicity, tumorigenic potential, and off-target effects
Comparison with established therapies such as autologous serum, platelet-rich plasma, topical cyclosporine …

Currently, translational challenges are mentioned but dispersed. Consolidation into a structured section would greatly improve impact.

5. Heterogeneity and Terminology

Although MISEV2023 is referenced, many cited studies likely did not fully comply with these updated standards.

The manuscript should critically address:
Whether EVs were adequately characterised in each study
Variability in isolation methods
Purity and contamination concerns
Differences between “exosomes” and mixed EV populations

This would strengthen scientific rigour and transparency.

6. Figures

A translational pipeline figure (bench → animal model → human trial → clinical implementation) would be valuable and would visually highlight the current gap between experimental promise and clinical application.

7. Critical Appraisal of Engineering Strategies

Numerous engineered exosome systems are described, including nanoparticle-modified vesicles, siRNA-loaded constructs, and hydrogel-based delivery platforms.

However, scalability and manufacturing feasibility are not critically evaluated. Some strategies, such as in situ nanocrystal growth or complex genetic engineering, may be difficult to translate into routine ophthalmic practice.

A brief feasibility analysis addressing cost, scalability, regulatory complexity, and industrial production would enhance realism.

8. Balance Between Mechanistic Detail and Clinical Relevance

The manuscript provides an extensive discussion of molecular pathways (NF-κB, MAPK, NLRP3, TLR4, Jak2/Stat3, etc.), which is scientifically valuable.

However, clinical implications are less emphasized. Some mechanistic descriptions could be condensed, with greater focus on:
Clinical significance
Magnitude of therapeutic effect
Comparison with existing treatments
Realistic therapeutic positioning

9. Comparative Perspective

The manuscript would benefit from a structured comparative paragraph addressing how exosome-based therapies compare with:
Autologous serum eye drops
Platelet-rich plasma
Topical cyclosporine or lifitegrast
Emerging biologic therapies

Such a comparison would contextualise their potential added value.

10. Limitations Section Needs Strengthening

The manuscript should explicitly state:
Lack of long-term safety data
Lack of large-animal translational models
Absence of randomised controlled human clinical trials
Uncertainty regarding optimal dose, administration route, and treatment frequency
Unknown durability of therapeutic effects

A clearly articulated limitations paragraph would significantly improve balance and credibility.

 

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript presents a thorough and well-balanced overview of the therapeutic role of exosomes in ocular surface diseases. The topic is highly relevant and aligns well with the journal’s focus on biomolecular mechanisms and translational applications. The review is well referenced and reflects a solid command of current literature.

To further enhance its impact and accessibility, the manuscript would benefit from some structural refinement and selective condensation. In particular, readability could be improved by streamlining certain sections and highlighting key concepts through visual summaries or tables.

Detailed suggestions are outlined below.

Major Points

The abstract is informative but somewhat dense. It would benefit from being streamlined to more clearly emphasize the main take-home messages, distinguishing between mechanistic insights, therapeutic applications, and future challenges.

Sections 2.1–2.3 provide detailed mechanistic information but would be greatly strengthened by a concise schematic summarizing the relationships among exosome biogenesis, molecular composition, and biological functions. Such a figure would significantly enhance reader comprehension.

Figures 2 and 3 are informative but would benefit from improved resolution and larger font sizes for readability.
It may also be helpful to include a summary table comparing exosome sources (e.g., MSC-derived, epithelial-derived, amniotic-derived), their cargo profiles, mechanisms of action, and therapeutic targets in OSDs.

The discussion section currently focuses mainly on summarizing existing studies. Its impact would be enhanced by including a more explicit critical appraisal of current limitations and challenges in exosome-based therapy, such as standardization, scalability, and regulatory considerations.

A brief forward-looking paragraph outlining ongoing clinical efforts or realistic translational timelines would also be valuable.

Minor Points

There are a few minor typographical errors (e.g., extra commas or spacing). Some long sentences could be divided to improve clarity.

The list is comprehensive but could be slightly streamlined by removing entries not directly used in the text.

Including very recent citations (2024–2025), particularly on engineered or targeted exosomes for ocular therapy, would further strengthen the manuscript’s currency.

This is a solid and valuable review with strong scientific content. With moderate structural refinement, clearer visual summaries, and a slightly more critical discussion, the manuscript will be significantly strengthened and well positioned for publication.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

1. This is a thorough and timely review on an important topic that is acquiring increasing interest in the research and clinical community. I applaud the authors on this interesting topic and comprehensive review.
2. In section “2.3. Exosome Isolation and Concentration”, please include key references for each of the commonly employed techniques listed in “2.3.1. Differential Ultracentrifugation (DUC)”, etc. For an entire page, page 6, of the review manuscript not a single reference is cited. Key references for each of these techniques should be added to the manuscript.
3. The review manuscript is relatively sparse on figures and tables, with only 3 figures, of which one is a flow diagram, and 0 tables. The manuscript would benefit from additional tables to synthesize the data along with more figures demonstrating the mechanism. The manuscript does not have any clinical photos of the diseases discussed. Tables could also help synthesize the topics presented.
4. The manuscript would benefit throughout by improving communication over what level of evidence the research that is described has been conducted in. Clinical trials vs ex vivo studies vs animal models, for example, should be clearly distinguished in the manuscript throughout it.
5. While the manuscript summarizes well the promise of the research, the lack of utilization in routine clinical practice should be discussed further and barriers to adoption should be discussed and addressed. While briefly mentioned in Section “7. Limitations and Future Directions”, this portion should be significantly lengthened and elaborated upon. Specifically, barriers to clinical adoption are not provided in sufficient detail.
6. The manuscript would benefit from more comparison of exosomes with competing technologies, such as the pros and cons of each and where exosomes shine along with areas that exosomes might not fare as well.
7. The manuscript would benefit from the authors’ perspective on future directions. While briefly mentioned in Section “7. Limitations and Future Directions”, this portion should be significantly lengthened and elaborated upon.
8. Please ensure that all references are put in square brackets. For example, on page 14, line 586, pathways108 appears to be a reference that is not in square brackets that should be updated to read “pathways[108].” This occurs again on page 14, line 592. “MSC-EVs109” should be updated to read “MSC-EVs[109]”.
9. Please check the manuscript for English spelling and grammar. For example. in Figure 1, “elidibility” should be updated to read “eligibility”. Page 4, line 124, “apoptsis” should be updated to read “apoptosis”.

Comments on the Quality of English Language

Please check the manuscript for English spelling and grammar. 

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

This review systematically summarizes the roles and therapeutic potential of exosomes in ocular surface diseases. It discusses the molecular mechanisms by which exosomes regulate inflammation, oxidative stress, immune responses, and tissue repair in conditions such as dry eye disease, corneal injury, and keratitis. The cited literature is generally up to date, and the discussion is well organized, with a clear logical structure linking molecular mechanisms to therapeutic applications. The article also evaluates preclinical evidence supporting exosome-based therapies and addresses current challenges for clinical translation, including standardization, large-scale production, and safety considerations.

 

However, I have some suggestions：

1. While clinical translation is briefly mentioned, the discussion lacks sufficient depth regarding key practical barriers, such as large-scale production, long-term safety, and delivery efficiency on the ocular surface. A more detailed and critical evaluation of these translational hurdles would enhance the manuscript’s relevance.
2. Although research on exosomes has expanded rapidly in recent years, their true therapeutic efficacy and underlying mechanisms remain incompletely understood. The authors should provide a more in-depth discussion of the current limitations associated with exosome-based therapies, including uncertainties regarding mechanisms of action, variability in therapeutic outcomes, long-term safety concerns, and translational feasibility. A clearer and more forward-looking perspective outlining future research directions would further strengthen the manuscript.
3. Exosomes also represent a promising platform for gene delivery applications, including the transport of therapeutic proteins and gene-editing tools such as CRISPR-based systems. The authors may consider expanding the discussion to address the potential of engineered exosomes as delivery vehicles for gene modulation strategies in ocular surface diseases. Including this emerging direction would broaden the scope of the review and enhance its forward-looking perspective.
4. The manuscript alternates between the terms “exosomes,” “extracellular vesicles (EVs),” and “small extracellular vesicles (sEVs).” The terminology could be standardized throughout the text to avoid potential confusion for readers.

ChatGPT is exclusively used for language polishing and enhancing the clarity of expression. 

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have appropriately addressed the suggestions and implemented all the requested changes, resulting in an improved manuscript. Therefore, it can be accepted in its current form.

 

Reviewer 2 Report

Comments and Suggestions for Authors

The revised version of the manuscript has been carefully evaluated. The authors have adequately addressed the concerns raised during the previous round of peer review. In particular, the revisions have improved the clarity of the manuscript, and the responses provided demonstrate that the authors have taken the reviewers’ comments into serious consideration.

The modifications introduced in the text and the additional clarifications provided throughout the manuscript have strengthened the overall quality of the review. The discussion of the mechanisms, therapeutic applications, and translational challenges of exosome-based therapies for ocular surface diseases is now clearer and more coherent.

Overall, the manuscript now presents a comprehensive and well-structured overview of the therapeutic potential of exosomes in ocular surface diseases. In my opinion, the authors have satisfactorily addressed the previous comments and the manuscript is suitable for publication in its current form.

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have performed a major revision and significantly improved the manuscript. All of the comments and recommendations of the reviewers were closely considered and appropriate changes were made in the manuscript to address the points raised by the reviewers with additional information and figures. These revisions have significantly improved the manuscript. The updated manuscript contributes significantly to the literature, and I applaud the authors for this significant work.