Review Reports - Engineered Liposomal Delivery of <i>Human ACE2</i> Across the Blood–Brain Barrier Attenuated Neurogenic Hypertension

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript addresses an important and timely problem: the delivery of ACE2 across the blood–brain barrier (BBB) for the treatment of neurogenic hypertension. The concept of dual-functionalized (Tf and Pen) liposomes is novel and relevant, especially given the clinical challenge of drug-resistant hypertension. The experiments are well-structured, progressing from in vitro characterization to in vivo efficacy. However, the text suffers from repetitive phrasing, occasional grammatical inconsistencies, and some awkward sentence structures that reduce clarity. Additionally, a few self-citations are either missing or incorrectly formatted.

Line 12–15:
Current: “Objective: To overcome this limitation, PEGylated liposomes were surface-modified with transferrin…”
Suggestion: Rephrase to a complete sentence: “The objective of this study was to overcome this limitation by modifying PEGylated liposomes with transferrin…”
Line 41–43:
Current: “This is partly due to the fact that about 15% of hypertension patients are resistant…”
Suggestion: Replace “due to the fact that” with “because” for conciseness.
Line 57–59:
Current: “to reduce sympathetic nervous activity for effectively treatment of resistant hypertension.”
Correction: “to reduce sympathetic nervous activity for the effective treatment of resistant hypertension.”
Line 64–65:
Current: “supported by substantial evidences.”
Correction: “supported by substantial evidence.”
Line 86–89:
Current: “evaluate this system transport efficacy across the BBB”
Correction: “evaluate the transport efficacy of this system across the BBB”
Line 124–127:
Clarify the ratio: “(125 μg Tf per μM phospholipid)” is ambiguous. Revise to “125 μg Tf per μmol phospholipid.”
Line 187–195:
The TEER and permeability equations are correct but phrased awkwardly. Suggest breaking into two sentences for readability.
Line 318–321:
Current: “sizes of plain liposomes ranged from 140 to 160 nm, and that surface modification… did not significantly alter particle size.”
Suggestion: Rephrase: “The sizes of plain liposomes ranged from 140 to 160 nm. Surface modification with Tf, Pen, or both did not significantly alter particle size.”
Line 450–462:
Current: “effects appearing five days after IV injection and persisting throughout the recording period.”
Correction: “with effects observed five days after IV injection and persisting throughout the recording period.”
Line 595–596:
Current: “neurovascular unit formation (or neurovascular coupling) in the EAPN BBB model contributes to the maintenance…”
Suggestion: Remove redundancy: “neurovascular unit formation in the EAPN BBB model contributes to maintaining…”
Line 627–632 (Discussion):
This section is strong but verbose. Suggest condensing: instead of “Systemic injection of Tf-Pen-Lip-pACE2 robustly increased ACE2 expression in PVN neurons, attenuated Ang II–induced neurogenic hypertension, reduced sympathetic hyperactivation, and corrected body fluid dysregulation,” → “Systemic Tf-Pen-Lip-pACE2 injection increased ACE2 expression in PVN neurons, attenuated Ang II–induced hypertension, and normalized sympathetic and fluid balance.”

Grammar

Consistent misuse of plural “evidences” (should be “evidence”).
Occasional missing articles (“in brain” → “in the brain”).
Overuse of “significantly” (appears >20 times). Consider variation.
Passive voice dominates; balance with active voice for clarity.

Self-Citations:

References [13], [25], [26] correctly cite previous work from the same group, but formatting varies (some abbreviations incomplete, e.g., Hypertension. 2007, 49, 926-31. → should be 926–931).
Ensure all prior studies from Sun et al. are cited consistently with journal guidelines (author initials, full page range, proper DOI format).
Cross-check that any methods “as previously described” (lines 110, 124, 187, etc.) correspond to actual references cited in the list.

Comments on the Quality of English Language

Consistent misuse of plural “evidences” (should be “evidence”).
Occasional missing articles (“in brain” → “in the brain”).
Overuse of “significantly” (appears >20 times). Consider variation.
Passive voice dominates; balance with active voice for clarity.

Author Response

Thank you for taking the time and effort to review our manuscript. We greatly appreciate your comments and recommendations, which have helped improve this work. Our responses to each question are listed below.

1) Line 12–15:
Current: “Objective: To overcome this limitation, PEGylated liposomes were surface-modified with transferrin…”
Suggestion: Rephrase to a complete sentence: “The objective of this study was to overcome this limitation by modifying PEGylated liposomes with transferrin…”

This suggestion has been incorporated. Thank you very much for your helpful feedback

2) Line 41–43:
Current: “This is partly due to the fact that about 15% of hypertension patients are resistant…”
Suggestion: Replace “due to the fact that” with “because” for conciseness.

Yes, this suggestion has been adapted. Thank you very much for your help.

3) Line 57–59:
Current: “to reduce sympathetic nervous activity for effectively treatment of resistant hypertension.”
Correction: “to reduce sympathetic nervous activity for the effective treatment of resistant hypertension.”

Thank you for pointing out this error. It has been corrected in the revised manuscript.

4) Line 64–65:
Current: “supported by substantial evidences.”
Correction: “supported by substantial evidence.”

Yes, this error has been corrected as suggested. We sincerely appreciate your careful observation.

5) Line 86–89:
Current: “evaluate this system transport efficacy across the BBB”
Correction: “evaluate the transport efficacy of this system across the BBB”

Yes, this error has been corrected in the revised manuscript. Thank you for your careful review.

6) Line 124–127:
Clarify the ratio: “(125 μg Tf per μM phospholipid)” is ambiguous. Revise to “125 μg Tf per μmol phospholipid.”

Yes, this error has been corrected as suggested. We sincerely appreciate your careful observation.

7) Line 187–195:
The TEER and permeability equations are correct but phrased awkwardly. Suggest breaking into two sentences for readability.

Yes, thank you for the suggestion. The description of this equation has been rephrased into two sentences, and the equation for the permeability coefficient has also been updated. We greatly appreciate your valuable feedback.

8) Line 318–321:
Current: “sizes of plain liposomes ranged from 140 to 160 nm, and that surface modification… did not significantly alter particle size.”
Suggestion: Rephrase: “The sizes of plain liposomes ranged from 140 to 160 nm. Surface modification with Tf, Pen, or both did not significantly alter particle size.”

Yes, this sentence has been revised in accordance with the suggestion. Thank you for your valuable feedback

9) Line 450–462:
Current: “effects appearing five days after IV injection and persisting throughout the recording period.”
Correction: “with effects observed five days after IV injection and persisting throughout the recording period.”

Yes, this sentence has been updated according to the recommendation. Thank you for your valuable feedback

10) Line 595–596:
Current: “neurovascular unit formation (or neurovascular coupling) in the EAPN BBB model contributes to the maintenance…”
Suggestion: Remove redundancy: “neurovascular unit formation in the EAPN BBB model contributes to maintaining…”

Yes, the “neurovascular coupling” has been removed in the revised manuscript. Thank you for your valuable suggestion

11) Line 627–632 (Discussion):
This section is strong but verbose. Suggest condensing: instead of “Systemic injection of Tf-Pen-Lip-pACE2 robustly increased ACE2 expression in PVN neurons, attenuated Ang II–induced neurogenic hypertension, reduced sympathetic hyperactivation, and corrected body fluid dysregulation,” → “Systemic Tf-Pen-Lip-pACE2 injection increased ACE2 expression in PVN neurons, attenuated Ang II–induced hypertension, and normalized sympathetic and fluid balance.”

Yes, this sentence has been updated according to the recommendation. We appreciate your valuable feedback.

12) Grammar

Consistent misuse of plural “evidences” (should be “evidence”). Corrected
Occasional missing articles (“in brain” → “in the brain”). Corrected
Overuse of “significantly” (appears >20 times). Consider variation. Some of them have been replaced by “markedly, remarkably, dramatically, and greatly”
Passive voice dominates; balance with active voice for clarity. Some passive sentences have been revised into active voice.

13) Self-Citations:

References [13], [25], [26] correctly cite previous work from the same group, but formatting varies (some abbreviations incomplete, e.g., Hypertension. 2007, 49, 926-31. → should be 926–931). Have been corrected
Ensure all prior studies from Sun et al. are cited consistently with journal guidelines (author initials, full page range, proper DOI format). Yes, all these citations have been checked
Cross-check that any methods “as previously described” (lines 110, 124, 187, etc.) correspond to actual references cited in the list. Yes, all these references have been checked

14) Comments on the Quality of English Language

Consistent misuse of plural “evidences” (should be “evidence”). Have been corrected
Occasional missing articles (“in brain” → “in the brain”). Have been updated
Overuse of “significantly” (appears >20 times). Consider variation. Have been revised
Passive voice dominates; balance with active voice for clarity. Some passive sentences have been revised into active voice

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The aim of this study is to design and evaluate PEGylated liposomes surface-modified with
transferrin (Tf) to facilitate Tf receptor binding at the BBB and conjugated with penetratin
(Pen), a cell-penetrating peptide, to enhance neuronal uptake. The manuscript is very interesting and well structured.

My comments are:

• A schematic presentation of liposomes should be added.
The aim of the study should be addressed clearly in the last paragraph of the introduction.
The role of each excipient.
Preformulation studies should be added.
Long-term stability of the prepared liposomes should be evaluated.
Biocompatibility of liposomes should also be evaluated.
The limitations of this formulation.

Author Response

We sincerely thank the reviewer for your time and effort in evaluating our manuscript. We greatly appreciate the positive feedback and constructive recommendations, which have contributed to improving the manuscript. Our detailed responses to each comment are provided below.

1) A schematic presentation of liposomes should be added.

Thank you for this insightful suggestion. A schematic presentation of liposomes has now been included in Figure 8.

2) The aim of the study should be addressed clearly in the last paragraph of the introduction.

Yes, the aim of the study has been clearly addressed in the last paragraph of the Introduction: “the present study aimed to characterize the Tf-Pen-liposome-mediated gene delivery system, evaluate the transport efficacy of this system across the BBB, and determine the effects of centrally delivering the ACE2 gene across the BBB using Tf-Pen-liposomes on neurogenic hypertension.”

3) The role of each excipient.

We appreciate this valuable suggestion. In the revised manuscript, the roles of all excipients, including phospholipids, cholesterol, and conjugates, have been thoroughly discussed in the Discussion section (page 16, lines 617-627).

4) Preformulation studies should be added.

We completely agree with the reviewer on the importance of preformulation studies, including chitosan-pDNA formulation, lipid selection and ratio, in vitro release, and cellular uptake. These studies have all been performed in our previous research, as documented in the following publications:

Lamptey RNL, Sun C, Singh J. Blood pressure reduction through brain delivery of nanoparticles loaded with plasmid DNA encoding angiotensin receptor shRNA. Mol Ther Nucleic Acids. 2024 May 8;35(2):102210. doi: 10.1016/j.omtn.2024.102210.

Sharma G, Modgil A, Sun C, Singh J. Grafting of cell-penetrating peptide to receptor-targeted liposomes improves their transfection efficiency and transport across blood-brain barrier model.J Pharm Sci. 2012 Jul;101(7):2468-78. doi: 10.1002/jps.23152.

Lakkadwala S, Dos Santos Rodrigues B, Sun C, Singh J. Biodistribution of TAT or QLPVM coupled to receptor targeted liposomes for delivery of anticancer therapeutics to brain in vitro and in vivo. Nanomedicine. 2020 Jan;23:102112. doi: 10.1016/j.nano.2019.102112.

5) Long-term stability of the prepared liposomes should be evaluated.

We completely agree with the reviewer on the importance of examining the long-term stability of the prepared liposomes. The time-dependent stability has been assessed in our previous studies, as documented in the following publication:

Sharma G, Modgil A, Layek B, Arora K, Sun C, Law B, Singh J. Cell penetrating peptide tethered bi-ligand liposomes for delivery to brain in vivo: Biodistribution and transfection. J Control Release. 2013 Apr 10;167(1):1-10. doi: 10.1016/j.jconrel.2013.01.016.

6) Biocompatibility of liposomes should also be evaluated.

We completely agree with the reviewer on the importance of assessing biocompatibility. In our previous studies, the effects of liposomes on the viability of endothelial cells, astrocytes, and neurons have been investigated, as detailed in the following publications:

Lakkadwala S, Dos Santos Rodrigues B, Sun C, Singh J. Dual functionalized liposomes for efficient co-delivery of anti-cancer chemotherapeutics for the treatment of glioblastoma. J Control Release. 2019 Aug 10;307:247-260. doi: 10.1016/j.jconrel.2019.06.033.

7) The limitations of this formulation.

One potential limitation of this liposomal formulation for brain delivery is its lack of regional specificity. Following systemic administration, the encapsulated cargo may distribute to multiple brain regions. Future studies could focus on developing brain region-specific liposomes through the conjugation of appropriate ligands.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The article is of significant interest, however, several modifications are needed. I recommend authors address the following.

Why did the authors use PEGylated lipid 2000? What was the reason for this fragment length? Were preliminary experiments conducted to understand what length of polymer chain to use and what amount of PEGylated lipid to incorporate into the membrane?
A similar question concerns the chitosan used. The authors indicate that "Chitosan was used to stabilize the pDNA complex, providing endosomal buffering, biodegradability, and low toxicity” but they don't explain why chitosan with this degree of polymerization was used."
It would be helpful if authors provide information on deacetylation degree of chitosan at the Materials and Methods section.
Electron micrographs of the liposomes used would be helpful.

Author Response

The article is of significant interest, however, several modifications are needed. I recommend authors address the following.

We sincerely thank the reviewer for dedicating time and effort to review our manuscript. We highly appreciate the constructive comments and suggestions, which have contributed to improving the manuscript. Detailed responses to each comment are provided below.

1) Why did the authors use PEGylated lipid 2000? What was the reason for this fragment length? Were preliminary experiments conducted to understand what length of polymer chain to use and what amount of PEGylated lipid to incorporate into the membrane?

Based on our previous studies, a PEG molecular weight of 2000 Da was chosen. Higher PEG weights enhance stealth properties and liposome flexibility but can reduce cellular uptake. In this study, a balance between these effects was considered. The interaction of PEGylated lipids with the cellular membrane has been assessed in our previous studies using fluorescence-labeled liposomes, as described in the following publication (plain liposome about 20%, Tf-liposome about 40%, dual liposome about 60%):

2) A similar question concerns the chitosan used. The authors indicate that "Chitosan was used to stabilize the pDNA complex, providing endosomal buffering, biodegradability, and low toxicity” but they don't explain why chitosan with this degree of polymerization was used."

Chitosan protects pDNA in gene delivery via several mechanisms: (1) its cationic nature allows complexation with negatively charged pDNA, protecting it from nuclease degradation; (2) the positive surface charge promotes interaction with the cell membrane, enhancing endocytosis; and (3) the proton sponge effect facilitates endosomal escape, preventing lysosomal degradation. The degree of deacetylation, molecular weight, and N/P ratio of chitosan were determined in our previous studies, as described in the following publication:

3) It would be helpful if authors provide information on deacetylation degree of chitosan at the Materials and Methods section.

A higher degree of deacetylation (DD) increases the number of amino groups, thereby enhancing the positive charge and electrostatic binding with pDNA (negative charge). Chitosan with an average molecular weight of 30 kDa and DD of 85–90% was used, which improves pDNA release and stabilizes the chitosan-pDNA complex. The information has been added to the Method section (Page 4, lines 142-144).

4) Electron micrographs of the liposomes used would be helpful.

We appreciate this valuable suggestion. An electron micrograph has now been included in Figure 2 of the revised manuscript.