Review Reports - miR-4516-Loaded Engineered Milk Extracellular Vesicles Attenuate Indoxyl Sulfate-Induced Mitochondrial Dysfunction and Improve Renal Function in a CKD Mouse Model

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

General comments:

This study presents an investigation into the use of engineered milk derived extracellular vesicles (EVs) for the targeted delivery of miR-4516 to treat chronic kidney disease (CKD). The research addresses a challenge in the field: the targeted delivery of miRNA therapeutics to both renal tubular and endothelial compartments. The data are robust, particularly the in vitro Seahorse analysis demonstrating the rescue of mitochondrial bioenergetics and the in vivo validation showing improved renal function indices. The combination of a biocompatible milk EV platform with surface modification (G3-C12/RGD) represents a promising strategy with translational potential.

However, while the phenotypic observations are compelling, several points regarding mechanistic depth and in vivo targeting validation require clarification and improvement before the manuscript should be considered. Therefore, major revisions are required before the manuscript can be considered for publication

Major comments:

The study shows that miR-4516 delivery improves mitochondrial function and reduces apoptosis, However, the current manuscript does not provide any molecular evidence that this specific pathway is engaged in the context of EV delivery. To strengthen the mechanistic claims, the authors should include Western blot and qPCR data showing the expression levels of key downstream targets in the treated cells or kidney tissues.
An initial safety assessment is necessary. The authors are encouraged to include basic histopathological analysis of major organs from the treated mice to rule out overt toxicity or inflammatory infiltration.

Minor comments:

Consider adding a schematic of the experimental workflow or an illustration to enhance readability.
In Figure 1A, the labeling for microRNA should be formatted with a hyphen for accuracy and consistency with standard nomenclature (i.e., miR-4516 instead of miR 4516).
The terms "in vitro" or "in vivo" throughout the text should be italicized.

Author Response

Comments and Suggestions for Authors

General comments:

Major comments:

The study shows that miR-4516 delivery improves mitochondrial function and reduces apoptosis, However, the current manuscript does not provide any molecular evidence that this specific pathway is engaged in the context of EV delivery. To strengthen the mechanistic claims, the authors should include Western blot and qPCR data showing the expression levels of key downstream targets in the treated cells or kidney tissues.

: We sincerely thank the reviewer for this important and constructive comment. We agree that the original manuscript primarily demonstrated the phenotypic effects of EV-mediated miR-4516 delivery on mitochondrial function and apoptosis, while the molecular evidence supporting pathway engagement was limited.

To address this concern, we added new molecular data to the revised manuscript. Specifically, we performed additional Western blot analyses in indoxyl sulfate-treated TH1 cells following treatment with miR-4516-loaded G3-C12/RGD-engineered EVs. Based on our previous findings and the mechanistic framework of the study, we examined the expression of SIAH3 and mitochondrial PINK1, which are key molecules associated with the miR-4516/SIAH3/PINK1-related mitochondrial regulatory pathway.

As shown in the newly added Figure 3A,B, indoxyl sulfate treatment was associated with increased SIAH3 expression and reduced mitochondrial PINK1 levels, whereas treatment with miR-4516-loaded engineered EVs attenuated SIAH3 expression and restored mitochondrial PINK1 expression. In addition, EV treatment improved mitochondrial complex I and complex IV activities under indoxyl sulfate stress conditions (Figure 3D,E). Collectively, these additional data provide molecular and functional support that EV-mediated miR-4516 delivery is associated with modulation of a mitochondrial regulatory pathway relevant to mitochondrial quality control under indoxyl sulfate stress conditions.

These new data have been incorporated into the revised manuscript in the Results section (Figure 3A–E) and are further discussed in the Discussion. The corresponding experimental procedures have also been added to the Materials and Methods section.

In addition, we revised the relevant statements throughout the manuscript to avoid overstating the mechanism. Accordingly, we now state that our findings support pathway modulation consistent with the miR-4516/SIAH3/PINK1-associated axis, rather than definitive confirmation of the entire signaling cascade.

We appreciate the reviewer’s helpful suggestion, which has strengthened the mechanistic interpretation of the revised manuscript.

An initial safety assessment is necessary. The authors are encouraged to include basic histopathological analysis of major organs from the treated mice to rule out overt toxicity or inflammatory infiltration.

: We sincerely thank the reviewer for this important and constructive suggestion. We agree that histopathological evaluation of major organs would further strengthen the initial safety assessment of the engineered EV platform.

However, we regret that we are unable to include these additional data in the current revision because the animal study had already been completed and no additional major-organ tissue samples from the same experimental cohort were available for further histopathological analysis. We acknowledge that the absence of major-organ histological evaluation is a limitation of the present study.

Accordingly, we have added this point to the Discussion as a study limitation. We now state that, although the current findings support the therapeutic potential of miR-4516-loaded G3-C12/RGD-engineered EVs, a more comprehensive safety evaluation, including major-organ histopathology and systemic toxicity assessment, will be required in future preclinical studies.

We appreciate the reviewer’s valuable suggestion, which has helped us improve the balance and rigor of the revised manuscript.

Minor comments:

Consider adding a schematic of the experimental workflow or an illustration to enhance readability.

: We sincerely thank the reviewer for this helpful suggestion. We agree that adding a schematic illustration of the experimental workflow would improve the readability of the manuscript and help readers better understand the overall study design. In response to this comment, we have added a schematic figure as Figure 7 in the revised manuscript. This figure provides an overview of the experimental workflow, including EV engineering, in vitro treatment, and in vivo evaluation, thereby facilitating a clearer understanding of the study strategy. We appreciate the reviewer’s valuable suggestion, which has improved the clarity and readability of the revised manuscript.

In Figure 1A, the labeling for microRNA should be formatted with a hyphen for accuracy and consistency with standard nomenclature (i.e., miR-4516 instead of miR 4516).

: We sincerely thank the reviewer for this careful observation. In response to the reviewer’s comment, we corrected the label in Figure 1A from “miR 4516” to “miR-4516” to ensure consistency with standard microRNA nomenclature. We appreciate the reviewer’s helpful suggestion

The terms "in vitro" or "in vivo" throughout the text should be italicized.

: We sincerely thank the reviewer for this careful comment. In response to the reviewer’s suggestion, we revised the manuscript and italicized in vitro and in vivo throughout the text in accordance with standard scientific formatting.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

Reviewer Report

The manuscript entitled “miR-4516-Loaded Engineered Milk Extracellular Vesicles Attenuate Indoxyl Sulfate–Induced Mitochondrial Dysfunction and Improve Renal Function in a CKD Mouse Model” presents an interesting and potentially impactful approach using engineered milk-derived extracellular vesicles (EVs) as a delivery platform for miRNA-based therapy in chronic kidney disease. The study combines in vitro mechanistic experiments with in vivo validation and demonstrates improvements in mitochondrial function, apoptosis, and renal biomarkers . The concept is timely and aligns with current interest in EV-based therapeutics; however, several critical methodological and conceptual issues need to be addressed to strengthen the rigor and translational relevance of the work.

In general, the manuscript is well organized and the data are coherent with the proposed hypothesis. The use of RGD-functionalization to enhance targeting and the focus on miR-4516 are scientifically justified. Nonetheless, the study does not fully meet current standards in extracellular vesicle research, particularly regarding isolation, characterization, and cargo validation.

First, regarding EV isolation, the authors used differential centrifugation followed by iodixanol density-gradient ultracentrifugation . While this approach is acceptable, the reporting does not comply with updated ISEV (MISEV) guidelines. Important details such as yield, reproducibility, and assessment of co-isolated contaminants from milk (e.g., casein or lipoproteins) are missing. Additional validation of purity, including protein-to-particle ratios and negative markers, is necessary to ensure that the preparation is not contaminated.

Second, the manuscript refers to the vesicles as “exosomes,” which is not sufficiently supported by the presented data. The reported size distribution (~150–200 nm) and limited marker analysis (CD9 and CD81 only) do not allow definitive classification. The authors should instead use the term “extracellular vesicles (EVs)” unless additional markers (e.g., TSG101, ALIX) and negative controls are provided. A more comprehensive characterization panel is required to confirm vesicle identity and purity.

Third, the sustainability and scalability of the milk EV platform are not adequately discussed. While milk is an attractive source, the manuscript should address batch variability, effects of processing (e.g., pasteurization), storage stability, and feasibility for large-scale production. These aspects are essential for translational application and should be discussed more critically.

Fourth, although the authors show increased levels of miR-4516 in EV preparations after loading , there is no clear evidence that the miRNA is encapsulated vesicles rather than externally associated. Key controls such as RNase protection assays (with and without detergent) are missing. Without these experiments, it is difficult to conclude that the EVs are functioning as true delivery vehicles rather than carriers of surface-bound RNA.

Finally, the reference list should be updated. The manuscript relies in part on older literature and appears to include substantial self-citation. The authors are encouraged to incorporate more recent studies (2025–2026) on milk-derived EVs, particularly those addressing isolation, heterogeneity, and therapeutic applications. In this context, it would also strengthen the manuscript to cite relevant work on the therapeutic potential of milk EVs, including Babaker et al. (Int J Mol Sci, 2022), along with more recent reviews and experimental studies.

In conclusion, the manuscript presents promising findings, but major revisions are required. Addressing the issues related to ISEV compliance, EV characterization, miRNA encapsulation, and literature updating will significantly improve the scientific rigor and impact of the study.

Comments on the Quality of English Language

none

Author Response

Reviewer Report

: We sincerely thank the reviewer for this important and constructive comment. We agree that detailed reporting of EV isolation, reproducibility, and purity is essential.
In the present study, milk-derived EVs were prepared using the same core isolation and purification workflow that we previously established for bovine milk EVs, consisting of sequential differential centrifugation followed by iodixanol density-gradient ultracentrifugation. In that previous study, the preparations were characterized by total protein measurement, nanoparticle tracking analysis for particle concentration and size distribution, and immunoblotting for the EV markers CD9 and CD81. In addition, after surface engineering and cargo loading, the EV preparations were re-isolated by iodixanol density-gradient ultracentrifugation to reduce carryover of unbound materials.
The current study employed the same core workflow, including density-gradient re-purification after G3-C12/RGD surface engineering and after miR-4516 loading. We have revised the Materials and Methods section to describe this procedure more explicitly and to cite the relevant previous study.
At the same time, we agree with the reviewer that broader purity assessment, including protein-to-particle ratio, negative or depleted markers, and direct evaluation of possible co-isolated milk contaminants such as casein or lipoproteins, would further strengthen the study. These analyses were not comprehensively included in the present work. We therefore acknowledge this as a limitation and have revised the manuscript to state that more extensive EV purity characterization will be required in future studies.
We appreciate the reviewer’s valuable comment.

Reference

Go, G.; Park, H.J.; Lee, J.H.; Yun, C.W.; Lee, S.H. Inhibitory effect of oxaliplatin-loaded engineered milk extracellular vesicles on tumor progression.Anticancer Res. 2022, 42, 857–866.

: We sincerely thank the reviewer for this important and constructive comment. We agree that the term “exosomes” should be used with caution when the available characterization data do not support definitive subtype classification. In response to the reviewer’s comment, we have revised the terminology throughout the manuscript and now consistently use “extracellular vesicles (EVs)” instead of “exosomes.” We agree that the size distribution observed in the present study, together with the limited marker analysis using CD9 and CD81, supports the use of the broader term “EVs” rather than definitive classification as exosomes.
We also acknowledge that a more comprehensive characterization panel, including additional positive markers such as TSG101 and ALIX as well as appropriate negative controls, would further strengthen vesicle characterization and purity assessment. However, these additional analyses were not comprehensively included in the present study. We therefore recognize this as a limitation of the current work and have reflected this point in the revised manuscript.
We appreciate the reviewer’s helpful comment, which has improved the accuracy and rigor of the manuscript.

: We sincerely thank the reviewer for this important and constructive comment. We agree that the sustainability and scalability of the milk-derived EV platform are important considerations for translational application and should be discussed more explicitly. In response to the reviewer’s comment, we revised the Discussion to address batch-to-batch variability, the possible effects of milk processing conditions such as pasteurization, storage stability, and the feasibility of large-scale production more specifically. We also clarified that source heterogeneity, processing conditions, and storage may influence EV yield, composition, and functional consistency, and therefore require further standardization of source material, purification workflow, storage conditions, and quality-control parameters for future preclinical and translational development. In addition, we emphasized that the present study should be regarded as a proof-of-concept evaluation of therapeutic potential rather than a fully optimized manufacturing platform. We appreciate the reviewer’s valuable comment.

: We sincerely thank the reviewer for this important comment. We agree that direct evidence distinguishing intravesicular encapsulation of miR-4516 from external association would further strengthen the interpretation of the loading data.
In the present study, miR-4516 was introduced into engineered milk-derived EV preparations using the Exo-Fect™ siRNA/miRNA transfection system, followed by iodixanol density-gradient ultracentrifugation to remove unincorporated miRNA and residual reagents. As shown in Figures 1A and 1E, RT–qPCR analysis demonstrated significantly increased miR-4516 levels in the re-purified EV preparations after loading. For this analysis, EV input was normalized by particle number determined by NTA, and cel-miR-39 was used as an exogenous spike-in control during RNA quantification. These results support increased association of miR-4516 with the re-purified EV preparations after loading.
However, we agree with the reviewer that these data do not definitively distinguish intravesicular encapsulation from externally associated miRNA. Because RNase protection assays in the presence and absence of detergent were not performed in the present study, we cannot make a definitive claim regarding the localization of miR-4516 within the EV preparations. Accordingly, we revised the manuscript to describe this point more cautiously and added it as a limitation in the Discussion. We further clarified that additional validation, including RNase protection assays with and without detergent, will be required in future studies to confirm the localization and protection status of the loaded miRNA. Therefore, the present data support EV-associated miR-4516 enrichment after loading, but do not by themselves establish intravesicular encapsulation or fully define the EVs as delivery vehicles in a strict mechanistic sense.
We appreciate the reviewer’s valuable comment.

: We sincerely thank the reviewer for this important and constructive comment. We agree that the reference list should be updated to better reflect recent progress in the field of milk-derived extracellular vesicles and to provide a broader and more balanced overview beyond our previous work. In response to the reviewer’s comment, we revised the reference list and updated the relevant sections of the Discussion to incorporate additional literature on milk-derived EVs, particularly studies addressing therapeutic potential, isolation and purity considerations, storage stability, and translational scalability. Specifically, we added the following references: [30] Babaker et al., Int. J. Mol. Sci. 2022; [31] Kong et al., Front. Pharmacol. 2025; [32] Dogan et al., J. Biol. Eng. 2025; and [33] Mata and Marcus, Nanotheranostics 2026. These references were incorporated to strengthen the discussion of therapeutic applications, isolation-related challenges, storage stability, and practical considerations for large-scale production of milk-derived EVs. In addition, we reviewed the reference list throughout the manuscript and incorporated more recent studies to reduce overreliance on older references and self-citation. We appreciate the reviewer’s valuable suggestion.

: We sincerely thank the reviewer for the careful evaluation of our manuscript and for the constructive summary comments. We appreciate the reviewer’s recognition of the significance of our findings.
In response to the reviewer’s major comments, we carefully revised the manuscript to improve its scientific rigor and clarity. Specifically, we revised the text to better align with current EV-related considerations, adopted more appropriate terminology for EV classification, expanded the discussion of EV characterization and purity-related limitations, clarified the interpretation of the miR-4516 loading data and acknowledged the lack of direct validation of intravesicular encapsulation, and updated the reference list with additional recent studies on milk-derived EVs.
We are grateful for the reviewer’s thoughtful suggestions, which helped improve the rigor and overall presentation of the manuscript.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have thoroughly revised the manuscript in response to the previous review comments.