Extracellular Vesicle-Associated miRNAs in Cornea Health and Disease: Diagnostic Potential and Therapeutic Implications

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors,

I have carefully reviewed your manuscript titled “Extracellular Vesicle-Associated miRNAs in Cornea Health and Disease: Diagnostic Potential and Therapeutic Implications.” The topic is highly relevant and timely, as EV-associated miRNAs are gaining importance in both diagnostics and therapeutics for ocular diseases. Your work provides a broad overview and collects valuable information; however, there are areas that need improvement in structure, consistency, and clarity before it can be considered for publication.

Major issues needs  corrections : 

1.  The heading “Corneal Disease Detection via EV-miRNA Signatures” appears more than once (section 6 at ~line 387 and again as section 7 at ~line 452). This creates literal duplication and confusion. Merge repeated material and renumber sections.
2. As a review, the manuscript should state how literature was selected (databases searched, keywords, date range, inclusion/exclusion criteria, whether PRISMA or narrative approach). I could not find a search-strategy or methods paragraph , please add a short “Search strategy” or “Methods” section e.g., databases, date limits, search terms, selection process, number of papers screened. This increases reproducibility and editorial rigor.
3. Inconsistent/contradictory size ranges for EV subtypes Text (section on EV subtypes) reports apoptotic bodies as 1–5 µm while Figure 2 caption lists apoptotic bodies as 500–2000 nm (0.5–2 µm). The exosome and microvesicle ranges also need to be consistent across text, figure captions and table(s). Please reconcile and pick authoritative ranges (cite MISEV guidelines) and correct all occurrences.
4. Table formatting esp. Table 1 & Table 3 Table 1 isolation methods   the table layout in the PDF appears uneven columns run together; headings truncated. Clarify units (e.g., throughput = samples/day?), add a short legend describing how you scored yield/purity/throughput (what do “High/Moderate/Low” mean?), and add references for each method entry.
5. Table 3 for each miRNA entry please consistently report species (human / mouse / rabbit), sample type (tear / aqueous / cell culture), validation method (qPCR / NGS), and whether functional validation was performed (yes/no + assay). Several entries lack species or context e.g., some entries have in vitro rabbit models vs. clinical human tears — make this explicit.
6. Lack of clarity on evidence level preclinical vs clinical Throughout the manuscript, it is sometimes unclear whether statements refer to in vitro, animal, or human clinical data. For translational claims (diagnostics/therapeutics), explicitly tag findings as preclinical/phase I/clinical case series. Readers need clear separation of evidence levels. Example: miRNA markers listed in Table 3 mix in vitro and clinical studies; label them.
7. Overstatements on clinical readiness While the technology is promising, some phrasing implies readiness for clinical use e.g., diagnostic “tear chip” in 30 minutes, translation to “predictive monitoring” without noting validation gaps and lack of standardized reference materials. Temper claims, add realistic milestones (analytical validation - clinical validation -prospective studies). Sections on regulatory roadblocks are good but expand them with proposed minimal evidence thresholds.
8. Figure 1 corneal cross sectio Caption OK as a schematic, but please ensure figure labels (epithelium, Bowman, stroma, Dua’s, Descemet’s, endothelium) are legible and correspond to text discussion of layer-specific miRNAs. Cite Fig 1 where you discuss layer-specific miRNAs in the text.
9. Figure 2 EV biogenesis / subtypes As noted, reconcile the size ranges for apoptotic bodies vs those listed in the text (one place says 1–5 μm; figure says 500–2000 nm). Use consistent units and add source (MISEV or primary review).
10. Figure 3 & Figure 4 diagnostic pipeline / therapy schematic These are conceptually useful. For Figure 3 (diagnostic pipeline) add an explicit box for “quality control / normalization” and state sample volumes expected (e.g., µL tears) if you refer to point-of-care. For Figure 4 : indicate loading efficiency ranges for different loading methods (passive incubation, electroporation, sonication) and add references.
11. Table 1 Reformat to MDPI style (simple columns, no merged cells across lines). Add a small footnote explaining how “Yield/Purity/Throughput” were classified. The current table is helpful but ambiguous.
12. Table 2 Good content. Please ensure each method has a one-line example application and limitations are succinct. Also ensure all references cited are present in the reference list (I spot many relevant citations, but cross-check).
13. Table 3 miRNA table nAs above: make species/sample/validation columns explicit, and consider adding a final column: “Translational status (preclinical/clinical)”. Several entries miR-328-3p, miR-4466, etc.need clearer notes on human vs animal origin.
14. Abbreviation typo: “CSSSC” is listed for Corneal Stromal Stem Cell in abbreviations; earlier text uses CSSC (single S). Please correct for consistency. (Abbreviations list, line ~658).
15. Ensure uniform reference formatting some entries look fine, but run a final check with your reference manager. Also ensure all in-text citations refer to entries in the reference list.
16. Redundant paragraphs about microfluidics + AI The microfluidics+AI concept appears repeatedly in similar wording. Consolidate into one focused subsection (diagnostics) and avoid verbatim repetition. See duplicated descriptions around lines ~295–320 and again ~420–438.

Comments on the Quality of English Language

The quality of English is generally understandable, but the manuscript would benefit from careful language polishing. There are several long and repetitive sentences, some awkward phrasing, and scattered typos/hyphenation errors. A thorough proofread or professional editing will help improve clarity, flow, and readability.

Author Response

Authors Response

Thank you for your thoughtful appraisal of our manuscript, “Extracellular Vesicle-Associated miRNAs in Cornea Health and Disease: Diagnostic Potential and Therapeutic Implications.” We appreciate your recognition of the topic’s relevance and timeliness. In response to your comments regarding structure, consistency, and clarity, we have undertaken a comprehensive revision. Below, we summarize the major changes and detail how each addresses your concerns.

Major issues needs corrections : 

1.  The heading “Corneal Disease Detection via EV-miRNA Signatures” appears more than once (section 6 at ~line 387 and again as section 7 at ~line 452). This creates literal duplication and confusion. Merge repeated material and renumber sections.

Authors Response

Thanks for pointing this out, authors much appreciated. Now, in the revised version of the manuscript, section 7 has been changed to “7 Therapeutic Implications of EV-Associated miRNAs” and highlighted.

 

2  As a review, the manuscript should state how literature was selected (databases searched, keywords, date range, inclusion/exclusion criteria, whether PRISMA or narrative approach). I could not find a search-strategy or methods paragraph , please add a short “Search strategy” or “Methods” section e.g., databases, date limits, search terms, selection process, number of papers screened. This increases reproducibility and editorial rigor.

Authors Response

Thank you for your thoughtful feedback. The authors have evaluated the review and confirm that it is narrative in nature, providing a comprehensive overview of the topic based on existing sources rather than following a systematic approach. Therefore, rigid search criteria typically required for meta-reviews and systematic reviews are not necessary in this context. However, in response to the reviewer’s suggestion, the authors have incorporated the search criteria into Section 2 (Methods) of the revised manuscript, with relevant changes clearly highlighted.

Recognizing the narrative rather than systematic nature of this review, we deliberately avoided rigid design filters and instead pursued a broad, inclusive search. We queried PubMed, EMBASE, Web of Science, Scopus, and Google Scholar for studies published between January 2000 and August 2025, using both controlled-vocabulary terms and free-text keywords “extracellular vesicle” or “exosome” combined with “microRNA” or “miRNA” and “cornea” or “corneal,” plus specific disorders (keratoconus; Fuchs endo-thelial corneal dystrophy; dry eye; limbal stem cell deficiency; alkali burn) and sup-plemented these with methodological and translational concepts (microfluidics; machine learning; artificial intelligence; Good Manufacturing Practices; regulatory frameworks). After deduplication, the authors screened titles and abstracts to identify original, peer-reviewed articles reporting EV isolation or characterization, miRNA profiling, functional assays, or preclinical/clinical corneal applications. We included Eng-lish-language primary research on EV-associated miRNAs in corneal health or disease, excluding reviews, editorials, conference abstracts, non-ocular studies, and papers lacking relevant miRNA data. Full texts of all eligible studies were then examined, with addi-tional hand-searching of pivotal journals and reference to the MISEV and EV-TRACK guidelines to ensure exhaustive coverage. This comprehensive methodology provided a robust and reproducible basis for integrating diagnostic, mechanistic, and therapeutic perspectives on EV-miRNAs in corneal disorders.

 

3 Inconsistent/contradictory size ranges for EV subtypes Text (section on EV subtypes) reports apoptotic bodies as 1–5 µm while Figure 2 caption lists apoptotic bodies as 500–2000 nm (0.5–2 µm). The exosome and microvesicle ranges also need to be consistent across text, figure captions and table(s). Please reconcile and pick authoritative ranges (cite MISEV guidelines) and correct all occurrences.

 

Authors Response

Thanks for your valuable comments. The authors have recognized those errors and have been corrected throughout the text and table as guided in the revised version of the manuscript, and highlighted.

 

Exosomes (30–150 nm), microvesicles (100–1000 nm), and apoptotic bodies (500–2000 nm)

 

4 Table formatting esp. Table 1 & Table 3 Table 1 isolation methods   the table layout in the PDF appears uneven columns run together; headings truncated. Clarify units (e.g., throughput = samples/day?), add a short legend describing how you scored yield/purity/throughput (what do “High/Moderate/Low” mean?), and add references for each method entry.

 

Authors Response

Thank you for raising this point. The authors have reviewed and not shown any formatting error from our end, and verified all tables 1, 2, and 3, confirming that they include appropriate references in indexed citations according to the journal template. These updates are reflected in the revised version of the review in the Word documents.

 

5 Table 3 for each miRNA entry please consistently report species (human / mouse / rabbit), sample type (tear / aqueous / cell culture), validation method (qPCR / NGS), and whether functional validation was performed (yes/no + assay). Several entries lack species or context e.g., some entries have in vitro rabbit models vs. clinical human tears — make this explicit.

 

Authors Response

 

Thank you for your constructive feedback. The authors have revised Table 3 to include each species and corresponding source tissues or cells for exosome extraction; in certain cases, entries reflect the presence of both studies, which accounts for two listings. All modifications are highlighted in the updated manuscript. Furthermore, the title and caption of Table 3 have been clarified to specify items in the table, such as cellular origin, pathological context, validated molecular targets or pathways, and key literature references, which are already present.

 

6 Lack of clarity on the evidence level, preclinical vs clinical. Throughout the manuscript, it is sometimes unclear whether statements refer to in vitro, animal, or human clinical data. For translational claims (diagnostics/therapeutics), explicitly tag findings as preclinical/phase I/clinical case series. Readers need a clear separation of evidence levels. Example: miRNA markers listed in Table 3 mix in vitro and clinical studies; label them.

 

Authors Response

Thank you for your feedback. The authors think and believe that the manuscript is intended to stimulate discussion through a holistic perspective, encompassing both preclinical and clinical aspects; however, it does not present diagnostic claims or therapeutic clinical data. Rather, it offers a narrative review and an overview of the potential applications of EV-miRNAs in disease diagnosis and therapeutic strategies, highlighting their precision by leveraging AI and ML approaches based on existing literature in the public domain. Section 5 focuses specifically on EV-associated miRNAs in the cornea, incorporating both clinical and preclinical material. Table 3, located within Section 5, provides a summary of all that includes the clinical and non-clinical miRNAs in the cornea and details species, source cells, targeted pathways, and references for each entry.  In Ophthalmology, cornea research is a niche field due to the anatomical challenges that limit the penetration of any treatment drug. This is why exosomes might be a good option, yet challenging, so there are very limited clinical studies available.

 

7 Overstatements on clinical readiness While the technology is promising, some phrasing implies readiness for clinical use e.g., diagnostic “tear chip” in 30 minutes, translation to “predictive monitoring” without noting validation gaps and lack of standardized reference materials. Temper claims, add realistic milestones (analytical validation - clinical validation -prospective studies). Sections on regulatory roadblocks are good but expand them with proposed minimal evidence thresholds.

 

Authors Response

 

Thank you for the comments. Upon review, it is noted that this manuscript is a narrative review, designed to present logical arguments and evidence from valid existing resources to address identified research gaps. Regarding the statement on "tear chip" in 30 minutes and its translation to "predictive monitoring," this is referenced in a previously published article (PMID: 35873535). The in-text citation and full reference are included in the bibliography. The reviewer is encouraged to review the cited source. Furthermore, section 9 covers roadblocks and breakthroughs, specifically focusing on challenges and limitations, including regulatory and technical issues, which are supported by appropriate references.

 

8 Figure 1 corneal cross sectio Caption OK as a schematic, but please ensure figure labels (epithelium, Bowman, stroma, Dua’s, Descemet’s, endothelium) are legible and correspond to text discussion of layer-specific miRNAs. Cite Fig 1 where you discuss layer-specific miRNAs in the text.

 

Authors Response

 

The authors appreciate the valuable feedback provided by the reviewers and have carefully examined the in-text citation for Figure 1. The placement of the Figure 1 citation has been verified to be correct within Section 3, and this adjustment is clearly highlighted in the revised version of the review.

“The cornea serves as the eye’s primary refractive interface and protective barrier, comprising anatomically distinct layers: the stratified squamous epithelium, Bowman’s layer, the collagen-rich stroma, Dua’s Layer, Descemet’s membrane, and the monolayer endothelium (Figure 1)”

 

9 Figure 2 EV biogenesis/subtypes As noted, reconcile the size ranges for apoptotic bodies vs those listed in the text (one place says 1–5 μm; figure says 500–2000 nm). Use consistent units and add source (MISEV or primary review).

 

Authors Response

The authors acknowledge this observation and, as recommended by the reviewer, have made the necessary corrections and updates in the revised manuscript.

Exosomes (30–150 nm), microvesicles (100–1000 nm), and apoptotic bodies (500–2000 nm)

 

10 Figure 3 & Figure 4 diagnostic pipeline/therapy schematic These are conceptually useful. For Figure 3 (diagnostic pipeline) add an explicit box for “quality control / normalization” and state sample volumes expected (e.g., µL tears) if you refer to point-of-care. For Figure 4 : indicate loading efficiency ranges for different loading methods (passive incubation, electroporation, sonication) and add references.

 

Authors Response

 

We appreciate the reviewer’s suggestion. We revised Figures 3 and 4 and their legends to incorporate the quality‑control elements in the diagnostic pipeline, loading efficiency ranges, and supporting references. The edits are highlighted in the revised version of the manuscript.

 

11 Table 1 Reformat to MDPI style (simple columns, no merged cells across lines). Add a small footnote explaining how “Yield/Purity/Throughput” were classified. The current table is helpful but ambiguous.

 

Authors Response

 

We appreciate the reviewer’s comments regarding Table 1. The authors have carefully reviewed the table to ensure it adheres to the journal’s style and requirements. Please note that Table 1 displays a comparative overview of conventional and emerging methods for exosome isolation, based on available resources. The meanings of Yield, Purity, and Throughput are clearly defined and self-explanatory; these metrics remain the primary means for characterizing the methods, and all these metrics largely vary depending on user output expectation, sample to sample, and method to method, so there are no strict criteria rather it is an output. For further details, we have provided appropriate references in each relevant row and column of the table. The authors believe that adding additional information could introduce unnecessary complexity and potentially distract readers, as such additions would be irrelevant in this context and unlikely to enhance the manuscript.

12 Table 2 Good content. Please ensure each method has a one-line example application and limitations are succinct. Also ensure all references cited are present in the reference list (I spot many relevant citations, but cross-check).

 

Authors Response

 

We thank the reviewer for the positive feedback on Table 2. In response, we have cross-checked the table to ensure that each method is accompanied by a concise, one-line example application to illustrate its practical use. We have also streamlined the “Limitations” column so that the descriptions are succinct and directly highlight the key drawbacks of each method. Furthermore, we carefully cross-checked all references cited in the table against the reference list and ensured that every citation is now included and formatted consistently. These revisions improve the clarity, utility, and accuracy of Table 2, making it more reader-friendly and aligned with the reviewer’s recommendations.

 

13 Table 3 miRNA table nAs above: make species/sample/validation columns explicit, and consider adding a final column: “Translational status (preclinical/clinical)”. Several entries miR-328-3p, miR-4466, etc.need clearer notes on human vs animal origin.

 

Authors Response

 

We thank the reviewer for these valuable suggestions to improve Table 3. In the revised version manuscript, we have made the species, source cell, disease association, and target pathways, which are the most essential things, to ensure clarity and consistency across all entries. In addition, we have added a new column titled “Translational Status (Preclinical/Clinical)” to indicate whether each reported miRNA finding has been validated in preclinical models or investigated in clinical/ patient-derived samples. For entries such as miR-328-3p and miR-4466, we have clarified the species of origin (human vs. animal). These refinements enhance the interpretability of the table and highlight the translational relevance of the reported miRNAs in corneal disorders.

 

14 Abbreviation typo: “CSSSC” is listed for Corneal Stromal Stem Cell in abbreviations; earlier text uses CSSC (single S). Please correct for consistency. (Abbreviations list, line ~658).

 

Authors Response

 

We thank the reviewer for pointing out this inconsistency. The abbreviation for Corneal Stromal Stem Cell has been corrected throughout the manuscript to CSSC (single “S”) for consistency. The error in the abbreviations list (line ~658) has also been fixed and highlighted in the revised version of the manuscript.

 

15 Ensure uniform reference formatting some entries look fine, but run a final check with your reference manager. Also ensure all in-text citations refer to entries in the reference list.

Authors Response

 

We appreciate the reviewer’s attention to detail. We have carefully re-checked all references using our reference manager to ensure uniform formatting throughout the manuscript. In addition, we verified that all in-text citations correspond accurately to entries in the reference list, and corrected minor inconsistencies where necessary. The reference section has now been standardized according to the journal’s style guidelines.

16 Redundant paragraphs about microfluidics + AI The microfluidics+AI concept appears repeatedly in similar wording. Consolidate into one focused subsection (diagnostics) and avoid verbatim repetition. See duplicated descriptions around lines ~295–320 and again ~420–438.

 

Authors Response

 

We thank the reviewer for identifying the redundant microfluidics + AI text and have consolidated the duplicated material into Section 7. Corneal Disease Detection via EV-miRNA Signatures, which is highlighted in the revised version of the manuscript.

 

Reviewer 2 Report

Comments and Suggestions for Authors

 Verma et al. in their manuscript “Extracellular Vesicle-Associated miRNAs in Cornea Health and Disease: Diagnostic Potential and Therapeutic Implications” intend to present a comprehensive review of extracellular vesicle–associated microRNAs (EV-miRNAs) in corneal biology, emphasizing their diagnostic potential and therapeutic applications. The review is timely, addresses a clinically relevant problem, and integrates interdisciplinary. This constitutes a comprehensive, and interesting topic of work that is appreciated. However, there are several Major concerns that can be resolved to improve the quality of the manuscript, listed below.

1. The Abstract is generally well written; however, it could be more impactful by reducing technical detail and emphasizing the key messages, particularly the diagnostic and therapeutic promise of EV-miRNAs and the main challenges ahead.
2. In the Introduction, the authors have provided a strong background, but the section is somewhat overly detailed regarding corneal disease burden and socioeconomic cost. Shortening this portion would be recommended so that the focus shifts more quickly toward EV-miRNAs.
3. At over 30 pages, the manuscript is lengthy and at times repetitive (for example, the EV isolation methods and AI/microfluidics discussions appear in multiple contexts). A more concise synthesis would significantly improve readability.
4. The review currently reads as more descriptive than critical. For example, while EV-miRNAs are presented as potential diagnostic biomarkers, the variability in published results (arising from patient heterogeneity or different isolation methods) should be explicitly contrasted and critically evaluated.
5. The balance between topics could be improved. Some sections (e.g., corneal architecture and general EV biology) are disproportionately detailed for a specialist readership, while important translational limitations (delivery efficiency, ocular clearance, off-target miRNA effects) are underexplored.
6. Stronger mechanistic integration is recommended. For instance, when discussing keratoconus miRNA signatures, please clarify how these intersect with biological processes such as ECM degradation, oxidative stress, or biomechanics.
7. In-text references to figures and tables are not always consistent or clearly explained. Please revise for clarity and ensure cross-referencing is accurate throughout.
8. The Conclusion is well written, but it could be strengthened by presenting clearer take-home messages and a roadmap that prioritizes future directions in this field.
9. Section 3 (EV Cargo Carriers in Action) is comprehensive, but it currently reads more like a methods textbook. Summarizing the isolation/characterization methods in a more concise way would improve flow.
10. Sections 4–6 (miRNA roles and diagnostics) are rich with examples. However, reorganizing the tables to emphasize the most clinically promising miRNAs (rather than providing long lists without sufficient context) would increase their impact.
11. Section 8 (Roadblocks) provides an impactful discussion of regulatory and technical challenges. This section would benefit from a clearer distinction between technical variability and regulatory gaps.
12. There are several minor typographical issues throughout the manuscript. For instance, “geted modulation”, “Off-tar- get effects” etc. In addition, spacing around citations is inconsistent in many places, such as “contamination challenges[85,198],” Careful proofreading will resolve these small but noticeable errors.
13. The formatting of abbreviations is not consistent across the text. For example, “iPSCs  and “MSC are presented differently; both should follow the same pluralization and abbreviation style. Similarly, hyphen usage is inconsistent, thoughout the manuscript .
14. Several sentences are overly long and complex, which affects readability. For instance, in the methods section describing EV isolation techniques, multiple methods are listed in one long sentence that could be divided into two for clarity. Breaking down such sentences will improve flow and accessibility for readers.
15. Some concepts are repeated in different sections. The requirement for strict safety evaluations is repeated in similar language. For a review paper, “compelling evidence” or “ultimately revolutionizing” may appear overstated. To make the flow more scholarly to readers, this language can be toned down.
16. Reference formatting is not consistent in terms of spacing and journal name styles. For example, “Br J Ophthalmol 2023, 107, 476-482” and “Cancers (Basel) 2021, 13” do not follow the same format. In addition, some tables contain broken words such as “Moder ate” and “concentratio n.” These should be addressed to improve readability.
17. Throughout the manuscript, some sentences are quite long and reduce clarity. Editing for conciseness and sentence flow is recommended.

Overall, this is an informative review that consolidates knowledge on EV-associated miRNAs in corneal health and disease and offers strong translational insights. To enhance its impact, we recommend reducing redundancy, sharpening the critical analysis, and improving figure/table referencing. With minor grammatical and formatting edits, this manuscript has the potential to make a significant contribution to the field.

 

Comments on the Quality of English Language

1. There are several minor typographical issues throughout the manuscript. For instance, “geted modulation”, “Off-tar- get effects” etc. In addition, spacing around citations is inconsistent in many places, such as “contamination challenges[85,198],” Careful proofreading will resolve these small but noticeable errors.
2. The formatting of abbreviations is not consistent across the text. For example, “iPSCs  and “MSC are presented differently; both should follow the same pluralization and abbreviation style. Similarly, hyphen usage is inconsistent, thoughout the manuscript .
3. Several sentences are overly long and complex, which affects readability. For instance, in the methods section describing EV isolation techniques, multiple methods are listed in one long sentence that could be divided into two for clarity. Breaking down such sentences will improve flow and accessibility for readers.
4. Some concepts are repeated in different sections. The requirement for strict safety evaluations is repeated in similar language. For a review paper, “compelling evidence” or “ultimately revolutionizing” may appear overstated. To make the flow more scholarly to readers, this language can be toned down.

Author Response

Authors Response

We are grateful for your positive assessment of our manuscript, “Extracellular Vesicle-Associated miRNAs in Cornea Health and Disease: Diagnostic Potential and Therapeutic Implications,” and for highlighting its timeliness, clinical relevance, and interdisciplinary integration. In response to your encouragement and to address the major concerns you noted, we have undertaken a comprehensive revision of the manuscript.

1.    The Abstract is generally well written; however, it could be more impactful by reducing technical detail and emphasizing the key messages, particularly the diagnostic and therapeutic promise of EV-miRNAs and the main challenges ahead.

Authors Response

We thank the reviewer for this constructive suggestion. In response, we have revised and highlighted in the revised version of the manuscript. Now, abstract reduces technical detail and places greater emphasis on the overarching messages. Specifically, we now highlight the diagnostic and therapeutic promise of EV‑miRNAs in corneal disease and clearly outline the main challenges that must be addressed for clinical translation. The revised abstract underscores the potential of EV‑miRNAs as non‑invasive biomarkers and therapeutic targets, while also drawing attention to the need for standardized methodologies and regulatory frameworks. We believe these changes improve the clarity, accessibility, and overall impact of the abstract.

Extracellular vesicle associated microRNAs (EV‑miRNAs) are emerging as pivotal regulators of corneal health and disease, holding exceptional promise for transforming both diagnostics and therapeutics. These vesicles carry distinct miRNA signatures in biofluids such as tears, offering a powerful, non‑invasive approach for early detection, risk stratification, and dynamic monitoring of corneal disorders. In addition, EV‑miRNAs act as key mediators of critical biological processes, including inflammation, fibrosis, and tissue repair. Consequently, they represent attractive therapeutic targets; for example, engineered EVs loaded with miRNA mimics or inhibitors can precisely modulate these pathways to promote regeneration and suppress disease progression. Yet, despite this considerable promise, the translation of EV‑miRNA research into clinical practice remains constrained by several challenges. Topmost among these are the lack of standardized EV isolation methods, variability in miRNA quantification, and the pressing need for regulatory frameworks tailored to the complexity of these biological therapeutics. Addressing these barriers is essential to ensure reproducibility, scalability, and safety in clinical applications. Accordingly, this review synthesizes current knowledge on EV‑miRNA profiles in corneal diseases, critically evaluates their diagnostic and therapeutic potential, and highlights strategies to overcome existing technical and regulatory limitations. Ultimately, the successful integration of EV‑miRNA-based approaches into personalized medicine frameworks could revolutionize the management of corneal diseases and substantially improve patient outcomes.

2.    In the Introduction, the authors have provided a strong background, but the section is somewhat overly detailed regarding corneal disease burden and socioeconomic cost. Shortening this portion would be recommended so that the focus shifts more quickly toward EV-miRNAs.

Authors Response

We appreciate the reviewer’s observation regarding the level of detail on corneal disease burden and socioeconomic cost in the Introduction. We respectfully note, however, that this section was intentionally written in short (only 196 words) to establish the clinical and societal significance of corneal disorders, thereby underscoring the urgent need for innovative diagnostic and therapeutic strategies such as EV‑miRNAs. By contextualizing the magnitude of disease burden and its economic implications, we aim to provide readers, particularly those less familiar with ophthalmic health disparities, with a clear rationale for why EV‑miRNAs represent a timely and impactful area of investigation. That said, we have carefully reviewed the section to ensure that the discussion remains concise and focused. While we retained the essential background to preserve its narrative value, we streamlined redundant details and refined transitions so that the focus shifts more smoothly toward EV‑miRNAs. We believe this balance maintains the strength of the introduction while addressing the reviewer’s concern.

3.    At over 30 pages, the manuscript is lengthy and at times repetitive (for example, the EV isolation methods and AI/microfluidics discussions appear in multiple contexts). A more concise synthesis would significantly improve readability.

Authors Response

We thank the reviewer for this valuable feedback. We would like to clarify that the manuscript contains 22 (content only) pages rather than 30. We acknowledge the concern regarding repetition, particularly in the sections on EV isolation methods and AI/microfluidics. In the revised version, we have carefully streamlined these discussions, consolidated overlapping AI/microfluidics content under section 7, and reduced redundancy to improve clarity and readability.

4.    The review currently reads as more descriptive than critical. For example, while EV-miRNAs are presented as potential diagnostic biomarkers, the variability in published results (arising from patient heterogeneity or different isolation methods) should be explicitly contrasted and critically evaluated.

Authors Response

We thank the reviewer for this insightful observation. We would like to clarify that the manuscript does address the variability in published results and the need for critical evaluation in Section 9: Roadblocks to Breakthroughs. In this section, we explicitly discuss how patient heterogeneity, differences in EV isolation methods, and variability in miRNA quantification contribute to inconsistent findings across studies. We also highlight these limitations as key barriers to clinical translation, thereby providing a critical perspective and identifying the research gap that must be addressed. To further strengthen this point, we have highlighted lines 539 to 575  in the revised version to make this critical evaluation more explicit and ensure that readers clearly recognize the challenges alongside the promise of EV‑miRNAs.

5.    The balance between topics could be improved. Some sections (e.g., corneal architecture and general EV biology) are disproportionately detailed for a specialist readership, while important translational limitations (delivery efficiency, ocular clearance, off-target miRNA effects) are underexplored.

Authors Response

We thank the reviewer for this thoughtful feedback. Ophthalmology is indeed a wide area of research with multiple tissues and disease contexts; however, this review specifically aims to focus on the mitigation of corneal pathologies via EV‑miRNAs. Accordingly, in Section 3: Corneal Architecture and Pathological Spectrum, we considered it essential to elaborate on corneal tissue anatomy and its subcellular counterparts. This background is within the scope of the review and provides readers with the necessary foundation to understand disease mechanisms before moving to therapeutic applications. With respect to translational limitations, we would like to clarify that delivery efficiency and ocular clearance are already discussed in Section 6: Tailored Extracellular Vesicles (TeVs) in Cornea (lines 365–377), while off‑target miRNA effects are addressed in Section 9: Roadblocks to Breakthroughs (lines 577–593). In the revised version, we have further highlighted these discussions to improve visibility and ensure that the balance between opportunities and limitations is clear to the reader.

 

6.    Stronger mechanistic integration is recommended. For instance, when discussing keratoconus miRNA signatures, please clarify how these intersect with biological processes such as ECM degradation, oxidative stress, or biomechanics.

Authors Response

We thank the reviewer for highlighting the need to more tightly integrate miRNA dysregulation with the key pathogenic pathways in keratoconus. In response, we have substantially revised the manuscript in lines 283- 306 to include a dedicated mechanistic view of it. Below is the newly added text and a brief overview of the changes

Mechanistically, the dysregulated miRNA landscape in keratoconus converges on three interlinked pathogenic axes: extracellular matrix (ECM) degradation, oxidative stress, and biomechanical dysfunction. Specifically, upregulated miR-143-3p, miR-182-5p, and miR-92a-3p enhance matrix metalloproteinase expression and directly repress structural collagens (e.g., COL1A1, COL4A4) and integrins (ITGA1/ITGA2), thereby accelerating stromal thinning and loss of corneal rigidity. Concurrently, downregulation of miR-29b-3p and miR-205-5p lifts inhibition on pro-fibrotic TGF-β signalling through derepression of SMAD2/3 targets and weakens collagen cross-linking, compounding ECM breakdown. Moreover, oxidative insults in KC epithelial and stromal cells provoke shifts in miR-151a-3p and miR-194-5p levels, which normally modulate antioxidant defence pathways via Nrf2 and superoxide dismutase 2. Their dysregulation tips the redox balance toward lipid peroxidation and mitochondrial dysfunction, further destabilizing the corneal microenvironment. Bioinformatic investigation has also uncovered a competitive endogenous RNA network featuring miR-4257 and miR-4494, which may govern posttranslational modifications and cellular stress adaptations in KC. Finally, aberrant biomechanical forces, whether from chronic eye rubbing or elevated intraocular pressure activate mechanotransducers YAP1/TEAD1 and TGF-β1, triggering a feedback loop in which cyclic stretch itself upregulates miR-21 and miR-34a. These miRNAs inhibit SMAD7 and other anti-fibrotic repressors, amplifying myofibroblast differentiation and protease release that propagate ECM loss and ectatic deformation. Together, these miRNA-driven processes form an integrated network: mechanical stress and oxidative damage reshape miRNA expression, which in turn dismantles ECM integrity and promotes fibrotic remodelling, offering precise mechanistic entry points for novel diagnostic biomarkers and targeted therapies in keratoconus

7.    In-text references to figures and tables are not always consistent or clearly explained. Please revise for clarity and ensure cross-referencing is accurate throughout.

Authors Response

We thank the reviewer for pointing this out. In the revised version, we have carefully reviewed all in‑text references and captions to figures and tables to ensure consistency and accuracy. Cross‑referencing has been corrected where needed, and explanatory text has been added or refined so that each figure and table is clearly introduced and contextualized within the main text. We believe these revisions improve clarity and readability throughout the manuscript.

8.    The Conclusion is well-written, but it could be strengthened by presenting clearer take-home messages and a roadmap that prioritizes future directions in this field.

Authors Response

We thank the reviewer for this valuable suggestion. However, we have already highlighted not only the conclusion section but also section 9, Roadblocks to Breakthroughs, the key take‑home messages regarding the diagnostic and therapeutic promise of EV‑miRNAs in corneal diseases. Additionally, we have incorporated food for thought, a clearer roadmap that prioritizes future directions, including the need for standardized methodologies, strategies to improve delivery efficiency and safety, and translational frameworks to bridge preclinical findings with clinical application. We believe these refinements make the Conclusion more impactful and provide readers with a forward‑looking perspective on the field.

9.    Section 3 (EV Cargo Carriers in Action) is comprehensive, but it currently reads more like a methods textbook. Summarizing the isolation/characterization methods in a more concise way would improve flow.

Authors Response

We thank the reviewer for this constructive suggestion. Section 3 (EV Cargo Carriers in Action) was designed to provide readers with a brief and comprehensive overview of the commonly used EV biogenies, isolation, and characterization methods. In the manuscript, we have already provided the methodological details of isolation and characterization methods. However, for the clear visibility to the reviewer, authors have highlighted the particular lines 209-231in the section.

10.    Sections 4–6 (miRNA roles and diagnostics) are rich with examples. However, reorganizing the tables to emphasize the most clinically promising miRNAs (rather than providing long lists without sufficient context) would increase their impact.

Authors Response

We thank the reviewer for this valuable suggestion. In the updated versions of the manuscript under Sections 5–7, indeed, a range of EV-miRNAs with cornea disease studies has been laid out, and we agree that reorganizing the tables to emphasize the most clinically promising miRNAs would enhance their impact. In the revised version, we have restructured Table 3 to highlight key miRNAs with Clinical status, whether miRNAs are associated with clinical study or preclinical therapeutic potential. However, due to anatomical challenges and a lack of standardization of EVs, there is a very low number of clinical studies available in the given context. Nonetheless, we believe these changes improve clarity, focus, and clinical relevance for the specialist readership.

11.    Section 8 (Roadblocks) provides an impactful discussion of regulatory and technical challenges. This section would benefit from a clearer distinction between technical variability and regulatory gaps.

Authors Response

We thank the reviewer for this constructive suggestion. Section 9 (Roadblocks to Breakthroughs) was designed to highlight not only both technical and regulatory challenges but also has miRNAs as an off-target risk and Quality Control, associated challenges, which have already been distinct from each other in separate paragraphs. In the revised version, we have explicitly highlighted to ensure that readers can better appreciate how methodological limitations differ from policy and regulatory barriers.

12.    There are several minor typographical issues throughout the manuscript. For instance, “geted modulation”, “Off-tar- get effects” etc. In addition, spacing around citations is inconsistent in many places, such as “contamination challenges [85,198],” Careful proofreading will resolve these small but noticeable errors.

Authors Response

We thank the reviewer for carefully noting these typographical and formatting issues. In the revised version, we have thoroughly proofread the manuscript to correct errors, and we have standardized spacing around all in‑text citations. These corrections have been implemented throughout the manuscript to ensure consistency, accuracy, and improved readability.

13.    The formatting of abbreviations is not consistent across the text. For example, “iPSCs  and “MSC are presented differently; both should follow the same pluralization and abbreviation style. Similarly, hyphen usage is inconsistent tthroughout the manuscript .

Authors Response

We thank the reviewer for carefully noting these inconsistencies. In the revised version, we have standardized the formatting of all abbreviations to ensure consistent pluralization and style. In addition, we have carefully reviewed and corrected hyphen usage throughout the manuscript to maintain consistency and adherence to journal style guidelines. These revisions improve the overall readability and presentation of the manuscript.

14.    Several sentences are overly long and complex, which affects readability. For instance, in the methods section describing EV isolation techniques, multiple methods are listed in one long sentence that could be divided into two for clarity. Breaking down such sentences will improve flow and accessibility for readers.

Authors Response

We thank the reviewer for this helpful observation. In the revised version, we have carefully edited these passages throughout the manuscript by breaking down long sentences into shorter, clearer statements and restructuring lists of methods for improved flow. These revisions enhance clarity and accessibility while preserving the necessary technical detail.

15.    Some concepts are repeated in different sections. The requirement for strict safety evaluations is repeated in similar language. For a review paper, “compelling evidence” or “ultimately revolutionizing” may appear overstated. To make the flow more scholarly to readers, this language can be toned down.

Authors Response
We thank the reviewer for this valuable observation. In the revised version, we have carefully reviewed the manuscript to reduce repetition and consolidated overlapping content to improve flow. In addition, we have refined the language to maintain a more scholarly and balanced tone appropriate for a review article. These revisions enhance readability and ensure a more precise and academic presentation.

16.    Reference formatting is not consistent in terms of spacing and journal name styles. For example, “Br J Ophthalmol 2023, 107, 476-482” and “Cancers (Basel) 2021, 13” do not follow the same format. In addition, some tables contain broken words such as “Moder ate” and “concentratio n.” These should be addressed to improve readability.

Authors Response

We thank the reviewer for carefully noting these formatting inconsistencies. In the revised version, we have standardized all reference entries to follow a uniform journal reference style. In addition, we have carefully proofread all tables to correct broken words that could cause a lack of alignment in the Word document. These revisions improve the overall readability and presentation quality of the manuscript.

17.    Throughout the manuscript, some sentences are quite long and reduce clarity. Editing for conciseness and sentence flow is recommended.

Authors Response

We thank the reviewer for this helpful suggestion. In the revised version, we have carefully edited the text for conciseness, breaking down complex or lengthy sentences into shorter, more direct statements. These revisions improve sentence flow, readability, and accessibility while preserving the intended meaning and technical accuracy.

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The revised version has been carefully checked and found to be satisfactory. All major and minor comments from the previous round have been appropriately addressed. Figures, tables, and section structures are corrected, and the new methods section adds clarity. The manuscript is now scientifically sound, well-organized, and clearly written. Only very minor editorial polishing may be done during copy-editing. I recommend the manuscript for acceptance in its current form.

Author Response

Thank you for the time and effort you dedicated to reviewing our manuscript. Your insightful and constructive comments have been invaluable in refining our analysis of extracellular vesicle associated miRNAs as biomarkers of corneal health and disease.

We greatly appreciate your thorough evaluation.

Reviewer 2 Report

Comments and Suggestions for Authors

Authors have addressed all the raised concerns and improved the manuscript accordingly. 

Author Response

Thank you for your valuable feedback and thoughtful suggestions. Your insights have significantly strengthened our manuscript. We appreciate the time and effort you dedicated to improving our work.