Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This article addresses a very interesting issue: comparing extracellular vesicles and vesicle-like particles produced by different kingdoms of organisms. The article focuses on comparing the effects of EVs on the mammalian immune system. Interest in extracellular vesicles as a therapeutic tool has steadily increased in recent years. Therefore, the search for vesicles with specific, targeted therapeutic potential remains an important and relevant task. However, it should be noted that this review is written in a rather general manner and does not delve into specific details. For example, vesicles obtained from different types of mammalian cells may have completely opposite potential. Therefore, the generalizations made by the authors in the article seem irrelevant. The authors should focus on a few cell types, for example, only immune cells. It would be interesting to compare the number/set of specific mediators carried by various vesicles (e.g., microRNA, DNA, etc.) and how the EV potential changes depending on their quantity. It is also recommended to emphasize the unique substances carried by EVs and their mechanisms of action. This is very well done for describing PAMPs in bacterial vesicles. Furthermore, the text should include a more specific description of the particles, cells, and animal models used in the cited studies.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

 This review establishes that extracellular vesicle origin is the primary determinant of therapeutic efficacy. Its novelty lies in the systematic "cross-kingdom" comparison, moving beyond single-source studies to provide a selection framework for clinical use. By linking biogenetic pathways to specific immune outcomes, the research demonstrates that while bacterial vesicles are ideal for potent vaccine adjuvants, plant-derived vesicles offer a uniquely low-immunogenic, stable platform for oral drug delivery and gene silencing applications.

Suggestions to author for how to improve the language/grammar while keeping the scientific meaning intact:

• The phrase "notably their interactions with and impacts on" is slightly repetitive; using "impact on" would be more concise.
• The word "shredding" in "shredding of the plasma membrane" is likely a misspelling or misuse of "shedding," which is the standard biological term for membrane vesicle release.
• The use of "strategic objectives" and "attack units" provides a personified, military-like tone to microbial biology, which is evocative but slightly non-standard for technical writing.
• There is a minor typo in "biogenesis can appear through more prominent cellular events", where "emerge" or "result from" might be more accurate than "appear."
• The phrase "although disagreeing results exist" is a bit awkward; "conflicting results" or "discrepant findings" would be more standard academic phrasing.
• There is a spelling error in the text: "bacteial in-fections" instead of "bacterial infections." Additionally, "Pathuclarly" is used instead of "Particularly.

Comments on scientific content:

• The article initially defines mammalian exosomes as typically having a diameter between "30 and 150 nanometers". However, in the discussion of plant-originated vesicles (PEVs), it refers to "exosome-like nanovesicles" as being in a range of "tens to low hundreds of nm". This inconsistency in size classification within the same text can lead to ambiguity regarding whether "exosome" is a size-defined or biogenesis-defined term. In established literature, the International Society for Extracellular Vesicles (ISEV) recommends the term "small EVs" (sEVs) for vesicles <200 nm, as "exosome" should strictly refer to those of endosomal origin regardless of slight size fluctuations. Reference: DOI: 10.1080/20013078.2018.1535750; PMID: 30637091
• In the introduction, the article states that "the size of EVs usually determines the information they carry". Later in the same section, it contradicts this by asserting that "biological origin of EVs is a critical determinant of their functional effects, even when their size remains consistent". This internal conflict leaves it unclear whether size or origin is the dominant factor in determining cargo and function. Most literature supports the latter, noting that the parent cell's physiological state and sorting machinery (biogenesis) are the primary drivers of cargo, not the physical dimensions of the vesicle itself. Reference: DOI: 10.1038/s12276-024-01209-y; PMID: 38553551
• "These findings support a framework in which EV origin determines immunological function and therapeutic applicability... selecting appropriate EV sources for vaccine development, regenerative medicine, and targeted delivery strategies". This general assertion regarding regenerative medicine and targeted delivery lacks specific recent examples of novel delivery methods or specialized cell therapies. Integrating PMID: 38029457 (Aglan et al., 2024) would provide a concrete, modern example of using stem cell-derived units specifically for diabetes cell therapy, while DOI: 10.1016/j.carbpol.2025.123773 (Mbituyimana et al., 2025) would strengthen the discussion on "targeted delivery strategies" by offering a sophisticated example of tree-thorn-inspired microneedles for long-term stability and effective tissue treatment.
• The article describes the formation of microvesicles as occurring through "direct outward budding and shredding of the plasma membrane". The use of the word "shredding" is scientifically incorrect and contradicts established biological terminology; the correct term is "shedding." Shredding implies a destructive, non-regulated fragmentation, whereas shedding (or blebbing) is a regulated, non-lethal process essential for maintaining cell viability during vesicle release. Reference: DOI: 10.1016/j.molcel.2012.01.003; PMID: 22305007
• In Section 3.1.1, the article describes the transfer of MHC-antigen complexes to other cells as a "mechanism known as 'cross-dressing'". However, it cites a paper (Zhang et al., 2008) that specifically discusses "Trogocytosis" in its title and findings. While related, trogocytosis involves the active "nibbling" and ingestion of membrane fragments between live cells, whereas cross-dressing specifically refers to the passive acquisition of pre-formed EVs by a recipient cell to display foreign MHC. The article conflates these distinct mechanisms. Reference: DOI: 10.1371/journal.pone.0003097; PMID: 18766224
• The text states that selective sorting of BEV cargo is a "precisely packaged" process that "serve[s] the bacterium's survival". It then notes that DNA typically enters vesicles during "explosive lysis". This is a scientific contradiction: a "selective" and "precise" packaging mechanism is incompatible with "explosive lysis," which is by definition a disruptive, stochastic event that reassembles membrane fragments from cell death. It cannot be both a strategically sorted process and a result of eruptive cell destruction simultaneously. Reference: DOI: 10.15252/embj.2021108174; PMID: 34327778
• The article claims that Outer-Inner Membrane Vesicles (OIMVs) in Gram-negative bacteria "have a structure similar to that of mammalian exosomes". This is scientifically incorrect. OIMVs are characterized by a double-bilayer structure (containing both inner and outer bacterial membranes), whereas mammalian exosomes are single-bilayer vesicles of endosomal origin. Their biogenesis and structural morphology are fundamentally different, making the comparison misleading. Reference: DOI: 10.1038/s41579-018-0112-2; PMID: 30405232
• "Plant-derived extracellular vesicles have garnered growing scholarly interest due to their capacity to interact with mammalian systems while exhibiting minimal intrinsic immunogenicity... [they are] promising candidates for the oral delivery of small RNAs and pharmaceutical agents". While the text highlights the potential for plant-derived and immunomodulatory agents, it lacks direct references linking these natural bioactive compounds to specific cancer or wound-healing pathways. Adding DOI: 10.21608/ejchem.2023.224011.8285 (Melegy et al., 2024) would substantiate the section on pharmaceutical delivery by providing data on the apoptotic impact of green tea polyphenols on cancer cells. Furthermore, DOI: 10.1007/s40200-025-01559-y (Zoheir et al., 2025) would provide the necessary scientific backing for claims regarding the "maintenance of epithelial barrier integrity" by demonstrating how bioactive acids improve wound healing through specific immunomodulatory and anti-inflammatory effects in diabetic models
• In the discussion of PEVs, the article states that "average plant EV preparations frequently appear larger and more heterogeneous than typical mammalian exosome isolates". However, it then immediately follows this by noting that "disagreeing results exist". While acknowledging conflicting data is good practice, the article fails to reconcile how it can simultaneously claim PEVs have the "broadest size heterogeneity" while later presenting them in Table 1 (not fully shown in snippets) as having a comparable or specific therapeutic profile. Reference: DOI: 10.3389/fcell.2025.1589550;
• In the author affiliations, the text refers to "Eötvös Lóránd University... H-1117 Budapes, Hungary". This is a typographical/geographical error. The correct spelling is "Budapest." While a minor language error, in the context of a formal research article peer review, such mistakes in the institutional address detract from the professional accuracy of the document.

Any papers recommended in the report are for reference only. They are not mandatory. You may cite and reference other papers related to this topic.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Dear authors,

This review compares extracellular vesicles from organisms belonging to different kingdoms: bacteria, plants, and animals. We describe the biogenesis of EVs, their composition, and some properties, in particular, their ability to regulate the immune response. We also provide insight into the potential clinical applications of EVs of different origins.

The introduction states that all EVs are "messages" sent by a cell to another cell, and that the meaning of the message depends on the vesicle's origin and its physical properties. The authors state that the goal of the review is to compare vesicles from different kingdoms. However, it is unclear why only mammals from the animal kingdom were selected. Many studies have examined the properties of EVs from frogs, worms, and other organisms in this kingdom. A clearer statement of purpose is needed. If a comprehensive comparison of the properties of EVs from different kingdoms is intended, the following aspects should be covered: the effects of EVs within a single organism (for example, how basil exosomes affect basil), the effects of EVs on organisms within a single kingdom (how basil exosomes affect fir tree cells), and the effects of EVs on organisms of another class. If the intention is only to highlight the effects of different EVs on the human immune system, this should be clearly stated. And in each specific case, the source of the EVs and the recipient cells should be specified (i.e., "host cells" is too general a term).

The use of terms describing EVs is described in detail in MISEV2023, which is not referenced in this review. Therefore, the EV classification used in this review is outdated.

Figure 1 does not correspond with the text. Specifically, the figure depicts microvesicle formation in plants, but this is not mentioned in the text. Instead, the text describes the formation of exocyst-positive vesicles, which are not shown in the figure. The same is true for bacterial cells. The depiction of the membranes of different bacteria does not correspond to their ultrastructure. The arrow leading from the early endosome to microvesicle formation raises questions. These processes are not related.

The review text is highly repetitive, poorly structured, and difficult to understand. It presents mostly well-known facts, described in general terms, without detail or clear diagrams.

Only one paragraph is devoted to problems and development prospects, which is extremely insufficient and does not provide a sufficient understanding of the authors' views on this issue.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

The manuscript reviews mammalian, bacterial, and plant-derived extracellular vesicles with a focus on immune functions and therapeutic applications. The topic fits Biomolecules, and the comparative scope is useful. However, the review remains largely descriptive and lacks sufficient critical analysis. Therefore, I recommend major revision.

1. The novelty and specific need for this review should be clarified more explicitly. Several recent reviews already discuss EV-mediated immune regulation [Buzás, E. I. The roles of extracellular vesicles in the immune system. Nature Reviews Immunology, 2023, 23, 236–250. doi 10.1038/s41577-022-00763-8] and [Kalluri, R. The biology and function of extracellular vesicles in immune response and immunity. Immunity, 2024, 57, 1752–1768. doi 10.1016/j.immuni.2024.07.009]. Bacterial membrane vesicles and OMVs have also been reviewed in detail [Gan, Y. et al. Bacterial membrane vesicles. Physiological roles, infection immunology, and applications. Advanced Science, 2023, 10, 2301357. doi 10.1002/advs.202301357] and [Peregrino, E. S. et al. The role of bacterial extracellular vesicles in the immune response to pathogens, and therapeutic opportunities. International Journal of Molecular Sciences, 2024, 25, 6210. doi 10.3390/ijms25116210]. Plant-derived EVs have also been covered previously [Lian, M. Q. et al. Plant-derived extracellular vesicles. Recent advancements and current challenges on their use for biomedical applications. Journal of Extracellular Vesicles, 2022, 11, e12283. doi 10.1002/jev2.12283]. The authors should therefore explain what this article adds beyond these existing reviews and why its comparative focus on mammalian, bacterial, and plant-derived EVs provides additional value for Biomolecules readers.

2. At present, mammalian EVs, bacterial EVs, and plant EVs are mostly summarized in separate sections, which limits the critical value of the review. The authors should directly compare how EV source affects cargo composition and immune behavior. Plant-derived EVs should be compared separately because their reported low immunogenicity and RNA-carrying capacity may favor oral delivery, but their composition varies substantially depending on plant source and isolation method. The authors should also discuss how these differences affect therapeutic suitability, including regenerative therapy for mammalian EVs, vaccine adjuvant use for bacterial OMVs, and mucosal delivery potential for plant-derived EVs.

3. The central idea that EV origin determines immunological function should be revised. EV function is influenced not only by biological origin, but also by parental cell state and culture conditions. Isolation protocol and vesicle subpopulation can also affect EV activity, while dose and recipient cell type may substantially alter immune outcomes.

4. Terms such as exosomes, microvesicles, apoptotic bodies, OMVs, and plant exosome-like vesicles should be used consistently. The authors should avoid calling vesicles “exosomes” unless their biogenesis has been demonstrated.

5. The discussion of plant-derived EVs should be revised to avoid overstatement. Claims regarding low immunogenicity, RNA-mediated interspecies communication, and therapeutic delivery potential are promising, but most supporting evidence remains preclinical. The authors should clearly distinguish well-established findings from proposed mechanisms or emerging hypotheses. They should also separate experimental potential from clinical readiness and discuss current limitations.

6. The therapeutic application section should clearly separate clinical evidence from in vitro and animal evidence. For mammalian EVs, bacterial OMVs, and plant-derived EVs, the authors should state which applications are supported by approved products, which are in clinical trials, and which remain experimental.

7. The bacterial EV section should better balance therapeutic potential and safety concerns. OMVs have strong adjuvant activity, but endotoxin content and reactogenicity remain important translational barriers. Batch variability and inflammatory toxicity should also be discussed more systematically.

8. Table 1 requires substantial revision. For example, “PRRs, TLRs, miRNAs” is not an appropriate description of a dominant immune pathway for bacterial EVs. The table should be redesigned to compare EV source, key molecular cargo, immune receptor or pathway, main immune effect, evidence level, and major limitation.

9. The figures should be improved for a review article. Figures 1 and 2 are useful as schematic summaries, but they remain too general. The authors should increase mechanistic clarity.

10. Several statements require clearer wording or correction. The manuscript contains repeated ideas, awkward phrasing, and unclear sentences. For example, parts of the introduction repeat the same point about origin and function.

11. The abbreviation list should be corrected. It currently includes generic template terms that are not relevant to this manuscript. The authors should remove irrelevant abbreviations and include only terms used in the article.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 5 Report

Comments and Suggestions for Authors

Specific comments are as follows:

1. The mechanistic-to-therapeutic connection needs to be strengthened. The manuscript describes the biogenesis, cargo composition, and immune effects of mammalian, bacterial, and plant-derived EVs, but the therapeutic section does not adequately connect these mechanisms to specific therapeutic strategies. For example, mammalian EVs are described as immunoregulatory and regenerative, bacterial OMVs as immunostimulatory and vaccine-related, and plant EVs as low-immunogenic oral delivery systems. However, the manuscript should more clearly explain why these mechanistic properties make each EV type suitable for particular disease settings. The authors should add a dedicated section or expanded discussion that directly maps EV origin, cargo, uptake mechanism, immune pathway, therapeutic indication, and key safety concerns.
2. The therapeutic application section is currently too general. The authors should include more concrete examples of how specific EV mechanisms support specific therapeutic applications,
3. Since natural EVs have heterogeneous cargo and variable potency, therapeutic development often requires engineering strategies, including surface ligand modification, immune-cell targeting, cargo loading, RNA or protein enrichment, and hybrid EV designs. The current manuscript mentions future engineering only briefly in the conclusion. The authors should refer more recent review (PMID: 39227240, 37686050), expand this discussion and explain how engineering can improve targeting, potency, cargo consistency, immune modulation, and safety
4. The authors should consider adding a summary table or figure comparing MEVs, BEVs/OMVs, and PEVs across therapeutic criteria, including immune effect, dominant cargo, preferred administration route, likely disease indication, engineering feasibility, safety concern, manufacturing challenge, and clinical maturity.
5. The authors should introduce recent literature on EV delivery platforms and translational barriers (e.g., 10.1038/s41551-026-01643-5, 10.1002/adhm.202301010), and better discuss how EV source, cargo, immune mechanisms, and engineering strategies connect to specific therapeutic applications.
6. Safety and standardization require more detailed discussion.
7. Table 1 should be expanded. In its current form, it summarizes cargo and immune effects, but it does not sufficiently connect these features to therapeutic applications, disease indications, or translational barriers.
8. Figure 2 could be improved by adding therapeutic implications next to each immune mechanism. For example, immunosuppression could be linked to inflammatory or regenerative therapy, while immune activation could be linked to vaccines or cancer immunotherapy.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The text of the article has been significantly improved in accordance with the reviewers' suggestions.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have addressed all comments

Reviewer 3 Report

Comments and Suggestions for Authors

Dear authors, thank you for the changes and clarifications. The corrected figures (1 and 2) are fully consistent with the text and facilitate its comprehension. Emphasizing the role of exosomes in immunomodulation certainly helps clarify the main idea of ​​the article. The data presented in the text now fully answer the questions posed in the introduction, and the text also describes the potential uses of vesicles in medicine. Therefore, the article can be published in its current form.

Reviewer 4 Report

Comments and Suggestions for Authors

The authors have addressed my comments, and I recommend acceptance in its present form.

Reviewer 5 Report

Comments and Suggestions for Authors

The authors have addressed my concerns.