Mannose-6-Phosphate-Tagged Liposomes Exhibit Increased Transcytosis Across Human Blood–Brain Barrier Model

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript evaluates M6P-functionalized liposomes for BBB transcytosis using a hiPSC-derived BMEC model. The work shows improved uptake, clathrin-dependent internalization, ligand-density-associated transport, intact particle recovery, and downstream delivery to neurons and astrocytoma cells. The study is timely and interesting, but receptor-level validation, quantitative clarity, translational relevance, and presentation require substantial improvement before publication in my view, overall.

Major comments

1. The manuscript presents M6P-liposomes as a receptor-mediated BBB transcytosis strategy, but the direct involvement of CI-M6PR is not yet sufficiently proven. The authors show CI-M6PR staining and use Pitstop 2 to implicate clathrin-mediated endocytosis, but this does not establish that the M6P receptor itself is responsible for uptake or transcytosis. The study would be much stronger if the authors included receptor-level validation, such as free M6P competition, CI-M6PR blocking antibody, receptor knockdown, or another suitable receptor-interference experiment. At minimum, the authors should improve the mechanistic language and clearly distinguish clathrin dependence from CI-M6PR-specific transport.
2. The ligand-density experiment is interesting, but the interpretation needs to be clarified. The authors report increasing transcytosis with increasing M6P content and describe a plateau around 10 mol%, yet the 25 mol% formulation appears to show a higher mean value than several intermediate formulations. The authors should more carefully explain whether the response truly plateaus, whether 25 mol% is statistically different from 10 to 20 mol%, and which formulation should be considered optimal. A clearer statistical comparison among ligand densities would improve the conclusion.
3. The comparison with transferrin is useful, but the claim that M6P-liposomes reach approximately 55% of transferrin-mediated transport needs more careful framing. Since transferrin-Cy3 is a soluble ligand and the M6P system is a liposomal nanoparticle, the comparison is informative but not perfectly equivalent. The authors should discuss this limitation and avoid overextending the comparison. The statement that M6P is “remarkably potent” should be moderated unless supported by more direct comparison with other nanoparticle systems under similar experimental conditions.
4. The authors should add a concise summary figure or graphical table that helps readers identify the major findings. This figure should summarize liposome composition, M6P/PEG ligand density, size, PDI, zeta potential, surface charge, comparator ligands, transcytosis efficiency, relative performance versus transferrin, and downstream uptake by neurons or astrocytoma cells. Such a figure would make it much easier for readers to understand the nanocarrier design, the materials involved, and the degree of success achieved by each formulation.
5. The manuscript would benefit from stronger translational context. The work demonstrates fluorescent liposome transport, but no therapeutic cargo is delivered and no functional biological effect is shown in downstream cells. Therefore, the authors should be cautious in presenting the system as a CNS therapeutic delivery platform. They should discuss what types of cargo could realistically be loaded, whether M6P decoration may alter cargo release or intracellular routing, and what additional in vivo or disease-model studies would be needed before therapeutic relevance can be claimed.
6. Although the authors report TEER/dextran controls and NTA-based evidence for intact particle recovery, these data should be presented in a way that more directly supports the main conclusion. The authors should ensure that barrier integrity data are shown for the key M6P and control formulations, especially after transcytosis assays, and should clarify the quantitative relationship between fluorescence-based transport and NTA-based particle counts. This would help readers judge whether fluorescence intensity reliably reflects transported liposome number rather than differences in labeling, recovery, or detection efficiency.
7. The change in TEER values with against days after the cell culture to mimic the BBB should be presented for an extended period of time, at least for 3 weeks or so (as required) to show the barrier formation progressed and the barrier layer integrity developed.

Minor comments

1. Several typographical errors require correction, including transferrin-mediaded, significaltly, transytosis, inhibiton, avergage, lysis the liposomes, and astrocytome.
2. The abstract is informative but overly compressed. The authors should revise it for clarity and avoid overstating the therapeutic significance before cargo-delivery data are available.
3. The manuscript should use consistent terminology throughout. The terms hiPSC-BMECs, hBMECs, hiPSC-hBMECs, Ct-PEG, Ct-liposomes, M6P-PEG, and M6P-liposomes should be standardized.
4. The figure legends are sometimes repeat methodological details already provided in the Methods section. They should be shortened and focused on what each figure represents along with the findings therein.
5. The authors should consistently define biological replicates. It should be clear whether n refers to independent differentiations, independent liposome preparations, transwell inserts, imaging fields, or individual cells.
6. The statistical reporting should be improved by adding exact p values where possible and specifying which post hoc tests were used after ANOVA.
7. The Discussion should be edited to separate data-supported conclusions from speculation. Statements about clinical promise, potency, and CNS-targeted therapy should be balanced with the limitations of an in vitro BBB model.

The manuscript contains a promising concept and useful experimental data, but stronger receptor-level validation, clearer quantitative presentation, more cautious interpretation, and improved organization are needed before publication.

Author Response

Reviewer 1:

This manuscript evaluates M6P-functionalized liposomes for BBB transcytosis using a hiPSC-derived BMEC model. The work shows improved uptake, clathrin-dependent internalization, ligand-density-associated transport, intact particle recovery, and downstream delivery to neurons and astrocytoma cells. The study is timely and interesting, but receptor-level validation, quantitative clarity, translational relevance, and presentation require substantial improvement before publication in my view, overall.

Major comments

1. The manuscript presents M6P-liposomes as a receptor-mediated BBB transcytosis strategy, but the direct involvement of CI-M6PR is not yet sufficiently proven. The authors show CI-M6PR staining and use Pitstop 2 to implicate clathrin-mediated endocytosis, but this does not establish that the M6P receptor itself is responsible for uptake or transcytosis. The study would be much stronger if the authors included receptor-level validation, such as free M6P competition, CI-M6PR blocking antibody, receptor knockdown, or another suitable receptor-interference experiment. At minimum, the authors should improve the mechanistic language and clearly distinguish clathrin dependence from CI-M6PR-specific transport.

1. Response:

We thank the reviewer for this important point and agree that our current data do not definitively demonstrate that CI-M6PR is the causal receptor responsible for M6P-liposome uptake and transcytosis across the BBB model. In the revised manuscript, we have therefore moderated the mechanistic language throughout. Our present data show that hiPSC-BMECs express CI-M6PR, that M6P-functionalized liposomes display greater uptake and transcytosis than matched control liposomes, and that this uptake is sensitive to Pitstop 2, supporting an M6P-dependent and clathrin-sensitive endocytic process.

In addition, our previous study showed that structurally related control ligands and similarly anionic ligands did not reproduce the uptake enhancement, that Pitstop 2 markedly reduced uptake of M6P-liposomes, and that free M6P partially inhibited internalization at high concentration. Together, these findings are consistent with participation of an M6P receptor pathway, but we agree that they do not on their own establish CI-M6PR-specific transport.

Accordingly, we now distinguish clearly between clathrin dependence and CI-M6PR specificity in the revised text. We also added a paragraph to the Discussion stating that definitive receptor assignment will require receptor-interference experiments such as free M6P or IGF2 competition, CI-M6PR blocking antibodies, or CI-M6PR knockdown/knockout, which are established approaches in the CI-M6PR field.

 

1. The ligand-density experiment is interesting, but the interpretation needs to be clarified. The authors report increasing transcytosis with increasing M6P content and describe a plateau around 10 mol%, yet the 25 mol% formulation appears to show a higher mean value than several intermediate formulations. The authors should more carefully explain whether the response truly plateaus, whether 25 mol% is statistically different from 10 to 20 mol%, and which formulation should be considered optimal. A clearer statistical comparison among ligand densities would improve the conclusion.

1. Response:
   We thank the Reviewer for this insightful observation regarding the ligand-density experiments. To clarify the interpretation of the transcytosis data, we have performed a more detailed statistical comparison between the formulations and revised the manuscript as follows:

• Statistical Plateau: While the mean value for the 25 mol% M6P formulation is numerically higher, our statistical analysis (one-way ANOVA followed by  Tukey’s post-hoc test) shows no significant difference (p > 0.05) between the 10, 15, 20, and 25 mol% groups. This lack of statistical significance indicates that increasing the ligand density beyond 10 mol% does not yield a further gain in transcytosis, supporting our description of a "plateau" effect.
• Optimal Formulation: Based on these results, we consider the 10-15 mol% formulation to be optimal. It represents the "saturation point" where maximum biological effect is achieved.
• Manuscript Revisions: We have updated Figure 4 and the Results section to include explicit p-values for these comparisons. We also added a sentence” higher mean values were observed at 25 mol%, do not represent a statistically superior performance over the 10 mol% formulation” in the results.

 

1. The comparison with transferrin is useful, but the claim that M6P-liposomes reach approximately 55% of transferrin-mediated transport needs more careful framing. Since transferrin-Cy3 is a soluble ligand and the M6P system is a liposomal nanoparticle, the comparison is informative but not perfectly equivalent. The authors should discuss this limitation and avoid overextending the comparison. The statement that M6P is “remarkably potent” should be moderated unless supported by more direct comparison with other nanoparticle systems under similar experimental conditions.

1. Response:

We thank the reviewer for this important point. We agree that the comparison between M6P-functionalized liposomes and transferrin-Cy3 should be interpreted with caution, as these systems are not directly equivalent. Transferrin-Cy3 is a soluble ligand that undergoes transferrin receptor-mediated transcytosis, whereas M6P-liposomes are nanoscale lipid vesicles whose transport is influenced not only by ligand–receptor interactions but also by particle size, surface composition, PEG density, membrane structure, and intracellular trafficking.

In the revised manuscript, we have therefore reframed transferrin-Cy3 as an internal positive control and benchmark for BBB transcytosis, rather than as a directly comparable delivery system. We have also moderated the language throughout the Results and Discussion. Specifically, we replaced statements such as “remarkably potent” with more cautious wording, stating that M6P-functionalized liposomes achieved a substantial level of transport relative to transferrin-Cy3 under our experimental conditions.

We now clarify that the approximately 55% value should be understood as a relative benchmark within this in vitro BBB model, not as evidence that M6P-liposomes are intrinsically equivalent or superior to transferrin-based systems.

We exchanged the paraph: “The ability of M6P liposomes to reach half the efficiency of transferrin suggests that M6P is a remarkably potent and effective ligand for mediating nanoparticle transport across cellular barriers. Its performance underscores the potential of glycan-based ligands to drive efficient and selective delivery, opening new avenues for targeted nanomedicine strategies, particularly in challenging biological environments such as the blood-brain barrier”

For: “Although M6P-functionalized liposomes reached approximately 55% of the transport observed for transferrin-Cy3 in this model, this comparison should be interpreted as an internal benchmark rather than a direct equivalence, since transferrin-Cy3 is a soluble ligand whereas M6P-liposomes are nanoscale vesicles whose transport depends on additional physicochemical and trafficking parameters.” In the revised version of the manuscript.

1. The authors should add a concise summary figure or graphical table that helps readers identify the major findings. This figure should summarize liposome composition, M6P/PEG ligand density, size, PDI, zeta potential, surface charge, comparator ligands, transcytosis efficiency, relative performance versus transferrin, and downstream uptake by neurons or astrocytoma cells. Such a figure would make it much easier for readers to understand the nanocarrier design, the materials involved, and the degree of success achieved by each formulation.

1. Response:

We thank the reviewer for this valuable suggestion. In response, we have added a concise graphical summary table to the revised manuscript that compiles the main physicochemical and biological findings of the study. The new figure summarizes liposome composition, M6P/PEG ligand density, particle size, PDI, zeta potential, comparator ligands, transcytosis efficiency, relative transport performance compared to transferrin, and downstream uptake by astrocytoma cells and hiPSC-derived neurons. We believe this addition substantially improves the accessibility of the manuscript and provides readers with a clearer overview of the nanocarrier design and the relative performance of each formulation.

1. The manuscript would benefit from stronger translational context. The work demonstrates fluorescent liposome transport, but no therapeutic cargo is delivered and no functional biological effect is shown in downstream cells. Therefore, the authors should be cautious in presenting the system as a CNS therapeutic delivery platform. They should discuss what types of cargo could realistically be loaded, whether M6P decoration may alter cargo release or intracellular routing, and what additional in vivo or disease-model studies would be needed before therapeutic relevance can be claimed.

1. Response:

We thank the reviewer for this important comment. We agree that the present study demonstrates transport of fluorescently labeled liposomes, but does not yet demonstrate therapeutic cargo delivery or functional efficacy in downstream target cells. We have therefore revised the manuscript to frame this work more cautiously as a proof-of-concept study for M6P-assisted BBB transport, rather than as direct evidence of therapeutic efficacy.

In the revised Discussion, we now clarify that M6P-functionalized liposomes could potentially be adapted for cargos compatible with lipid-based nanocarriers, including small molecules, peptides, proteins/enzymes, and nucleic acids. However, we also acknowledge that cargo loading, release kinetics, endosomal/lysosomal routing, and intracellular bioavailability will need to be evaluated for each therapeutic payload. This is particularly relevant for M6P-decorated systems, as M6P may favor trafficking toward endosomal or lysosomal compartments, which could be advantageous for lysosomal enzyme delivery but less suitable for cargos requiring cytosolic release.

We have also added a limitation stating that therapeutic relevance will require further validation in disease-relevant models, including cargo-loaded liposomes, functional rescue assays in target cells, in vivo biodistribution, brain exposure studies, safety assessment, and efficacy testing in appropriate CNS or lysosomal storage disease models.

We replace the paraph in the Conclusion section: “Collectively, our results validate M6P as a functional ligand that enables both enhanced uptake and transcytosis across a human-relevant BBB model in vitro without compromising barrier integrity. This approach offers a CNS-targeting mechanism distinct from more broadly expressed receptors, such as the transferrin receptor, potentially reducing peripheral off-target effects. The use of hiPSC-derived BBB and neuronal models also enhances translational relevance and allows for future patient-specific therapeutic testing.”

 For the following: “While the present study establishes M6P-functionalization as a strategy to enhance liposome uptake and transcytosis across a human in vitro BBB model, it does not yet demonstrate delivery of a therapeutic cargo or functional rescue in downstream cells. Future studies should evaluate cargo-loaded M6P-liposomes, including small molecules, proteins/enzymes, or nucleic acids, and determine whether M6P decoration influences intracellular routing, cargo release, and bioavailability. Given the known role of M6P in lysosomal trafficking, this strategy may be particularly suitable for cargos intended for endosomal or lysosomal delivery, although it may require additional formulation optimization for cargos requiring cytosolic release. In vivo biodistribution, brain penetration, safety, and disease-model efficacy studies will be required before therapeutic relevance can be established”.

 

1. Although the authors report TEER/dextran controls and NTA-based evidence for intact particle recovery, these data should be presented in a way that more directly supports the main conclusion. The authors should ensure that barrier integrity data are shown for the key M6P and control formulations, especially after transcytosis assays, and should clarify the quantitative relationship between fluorescence-based transport and NTA-based particle counts. This would help readers judge whether fluorescence intensity reliably reflects transported liposome number rather than differences in labeling, recovery, or detection efficiency.

1. Response:
   We appreciate the Reviewer’s suggestion to integrate the barrier integrity and NTA data more directly. We have addressed these points as follows:

• Barrier Integrity (TEER): We have updated the figures where transcytosis was measured (Figure 4, 5 and 6) to include the TEER values recorded specifically at the end of each experiment. These data demonstrate that the barrier integrity remained intact and was not compromised by the treatment with either M6P or control formulations.
• Fluorescence vs. Particle Count (NTA): To ensure that fluorescence intensity reliably reflects the number of transported liposomes, we have clarified our quantification protocol in the Materials and Methods section:

• NTA was used to establish a correlation between liposomes integrity and fluorescence signal and not for liposomes quantification.
• To avoid signal saturation and maintain measurements within the linear dynamic range of the detector, specific dilution factors were applied: 1:400 for control (Ct) liposomes and 1:10,000 for M6P liposomes.

1. The change in TEER values with against days after the cell culture to mimic the BBB should be presented for an extended period of time, at least for 3 weeks or so (as required) to show the barrier formation progressed and the barrier layer integrity developed.

1. Response:

We thank the reviewer for this comment. We agree that long-term monitoring of barrier integrity can provide useful information regarding the stability of in vitro BBB models. However, in the context of hiPSC-derived BMEC systems, extended culture periods of 3 weeks are not generally required to demonstrate functional BBB maturation and are not standard practice in the field.

Our differentiation protocol follows and adapts previously established hiPSC-BMEC methodologies, in which barrier maturation is typically achieved within 7–10 days after induction and shortly after subculture onto transwell inserts. Multiple studies have shown that hiPSC-derived BBB models reach maximal TEER values during this period, after which TEER frequently plateaus or gradually declines due to prolonged culture stress and phenotypic drift rather than continued maturation. For example, Lippmann et al. and subsequent optimized Wnt-activated protocols reported functional BBB formation within approximately 8–10 days, with TEER values ranging from 1000 to >4000 Ω·cm² during this window [20–24, 28–32].

In our system, TEER progressively increased throughout differentiation and reached approximately 4000 Ω·cm² by day 10, together with minimal permeability to 70 kDa dextran (~0.3% transport over 6 h), indicating formation of a highly restrictive endothelial barrier. Importantly, all uptake and transcytosis experiments were performed during this established high-TEER window, and TEER values remained stable during the experimental time course, indicating preserved barrier integrity.

 

Minor comments

1. Several typographical errors require correction, including transferrin-mediaded, significaltly, transytosis, inhibiton, avergage, lysis the liposomes, and astrocytome.

We thank the reviewer for carefully identifying these typographical and spelling errors. All noted mistakes, have been corrected throughout the revised manuscript.

1. The abstract is informative but overly compressed. The authors should revise it for clarity and avoid overstating the therapeutic significance before cargo-delivery data are available.

We thank the reviewer for this helpful suggestion. The abstract has been revised to improve clarity, readability, and flow. We also moderated statements regarding therapeutic applicability to better reflect the proof-of-concept nature of the current study.

1. The manuscript should use consistent terminology throughout. The terms hiPSC-BMECs, hBMECs, hiPSC-hBMECs, Ct-PEG, Ct-liposomes, M6P-PEG, and M6P-liposomes should be standardized.

We thank the reviewer for this observation. In the revised manuscript, we standardized the terminology throughout the text, figures, and figure legends to improve clarity and consistency. Specifically, the terms referring to the endothelial model were unified as “hiPSC-BMECs,” while liposome formulations were consistently referred to as “Ct” liposomes and “M6P” liposomes.

1. The figure legends are sometimes repeat methodological details already provided in the Methods section. They should be shortened and focused on what each figure represents along with the findings therein.
2. We thank the reviewer for this helpful suggestion. In the revised manuscript, the figure legends were carefully edited and shortened to improve readability and avoid unnecessary repetition of methodological details already described in the Methods section.
3. The authors should consistently define biological replicates. It should be clear whether n refers to independent differentiations, independent liposome preparations, transwell inserts, imaging fields, or individual cells.

We thank the reviewer for this important comment. In the revised manuscript, we clarified the definition of biological replicates throughout the Methods section, figure legends, and Results. Specifically, we now indicate whether “n” refers to independent hiPSC differentiations, independent liposome preparations, transwell inserts, or analyzed cells, depending on the experiment. This information has been added systematically to improve transparency and reproducibility of the reported data.

1. The statistical reporting should be improved by adding exact p values where possible and specifying which post hoc tests were used after ANOVA.

We thank the reviewer for this valuable suggestion. In the revised manuscript, statistical reporting was improved. Exact p values were added whenever possible in each figure and the statistical methods section now clearly specifies the post hoc tests used following ANOVA analyses.

1. The Discussion should be edited to separate data-supported conclusions from speculation. Statements about clinical promise, potency, and CNS-targeted therapy should be balanced with the limitations of an in vitro BBB model.

We thank the reviewer for this important comment. In the revised manuscript, the Discussion was carefully edited to more clearly distinguish data-supported conclusions from forward-looking interpretations and speculation. Statements regarding clinical promise, potency, and CNS-targeted therapeutic applications were moderated throughout the text to better reflect the proof-of-concept nature of the present study and the limitations inherent to an in vitro BBB model.

The manuscript contains a promising concept and useful experimental data, but stronger receptor-level validation, clearer quantitative presentation, more cautious interpretation, and improved organization are needed before publication.

Reviewer 2 Report

Comments and Suggestions for Authors

In order to create an in vitro blood-brain barrier model that is physiologically appropriate, Margarita C. Dinamarca et al. activated canonical Wnt/β-catenin signaling in traditional differentiation methods for hiPSC-derived human brain microvascular endothelial cells. The scientists employed this model to examine the transcytosis efficacy of M6P-functionalized liposomes, which have been demonstrated in earlier research to show increased uptake in a variety of cell lines because of the presence of M6P on their surface.

The findings of the study confirm that M6P is a functional ligand that permits improved absorption and transcytosis across a human-relevant BBB model in vitro without sacrificing barrier integrity. This strategy may lessen peripheral off-target effects by providing a CNS-targeting mechanism different from more widely expressed receptors like the transferrin receptor. Future patient-specific treatment testing is made possible by the use of hiPSC-derived BBB and neuronal models, which also improves translational relevance.

 

 

The writing style is superb and the subject matter is highly captivating.
The current version of the manuscript is acceptable. Nonetheless, it will be helpful to include a small paragraph in the conclusion section comparing the results of the submitted research to those of an earlier study conducted by the authors (Reference 25).

 

Author Response

Reviewer 2

In order to create an in vitro blood-brain barrier model that is physiologically appropriate, Margarita C. Dinamarca et al. activated canonical Wnt/β-catenin signaling in traditional differentiation methods for hiPSC-derived human brain microvascular endothelial cells. The scientists employed this model to examine the transcytosis efficacy of M6P-functionalized liposomes, which have been demonstrated in earlier research to show increased uptake in a variety of cell lines because of the presence of M6P on their surface.

The findings of the study confirm that M6P is a functional ligand that permits improved absorption and transcytosis across a human-relevant BBB model in vitro without sacrificing barrier integrity. This strategy may lessen peripheral off-target effects by providing a CNS-targeting mechanism different from more widely expressed receptors like the transferrin receptor. Future patient-specific treatment testing is made possible by the use of hiPSC-derived BBB and neuronal models, which also improves translational relevance.

 

The writing style is superb and the subject matter is highly captivating.
The current version of the manuscript is acceptable. Nonetheless, it will be helpful to include a small paragraph in the conclusion section comparing the results of the submitted research to those of an earlier study conducted by the authors (Reference 25).

Response:

We thank the reviewer for the positive evaluation of our manuscript and for the helpful suggestion. In response, we have added a paragraph to the Conclusion section comparing the present findings with our previous study (Reference 25):

“Our findings build upon our previous work demonstrating that M6P-functionalized liposomes exhibit enhanced cellular uptake across multiple cell types compared with structurally related control formulations [25]. In that study, M6P-mediated uptake was shown to be ligand-specific, as neither similarly anionic liposomes nor structurally related glycans reproduced the effect. Moreover, uptake of M6P was markedly reduced by Pitstop 2 treatment, supporting the involvement of a clathrin-associated endocytic pathway. Free M6P competition experiments also partially reduced liposome internalization, suggesting participation of an M6P-sensitive receptor pathway, although not definitively establishing CI-M6PR-specific causality. Together with the current data, these observations support the conclusion that M6P-functionalization promotes ligand-dependent and clathrin-sensitive uptake and transcytosis across hiPSC-derived BMECs. Importantly, the present study extends these earlier findings by demonstrating that enhanced uptake is associated with increased transport across a physiologically relevant human BBB model and improved downstream delivery to neurons and astrocytoma cells. Although CI-M6PR represents a biologically plausible mediator based on its known role in M6P trafficking and clathrin-associated internalization, the current data do not exclude the possibility that additional glycan-sensitive uptake mechanisms contribute to the observed phenotype. Future studies employing receptor-interference approaches, including free M6P or IGF2 competition, receptor-blocking antibodies, or CI-M6PR knockdown/knockout models, will therefore be important to establish receptor-specific causality more definitively.”

Reviewer 3 Report

Comments and Suggestions for Authors

This is a very interesting and well-designed manuscript. However, a few queries need to be answered.

 

1. The data in Fig 1D are from only hBMECs. What is the expression level of different proteins in hiPSC? How do you compare the difference in expression levels?

 

2. The same question applied to the data presented in Supplementary Figure 1.

 

3. Please label Fig 1F more clearly, possibly with a different colour code.
4. The idea of using M6P in conjugation is excellent. Is it possible that D-mannose or M1P conjugation will show a similar effect?

 

5. From Fig 2A, it is unclear what the structural features of the liposomes are. What is the composition in the core and in the outer layer? Show the structure of PEG-conjugated M6P.

 

6. The data in Fig 3A do not show a significant increase in Fluorescence. Is the fluorescent marker stable inside the cells? What is the uptake efficiency of the naked FL dye?

 

7. The values of FL intensity (Fig 3E) may provide a better visibility if provided in a table with raw values.

 

8. Similarly, some of the values presented in Fig. 4 can be put together in a table.

 

9. The data showed in Fig6D does not a significantly increase uptake. If an FL dye is used for a biomarker, the change in uptake level or the difference should be many more folds than shown here. What is the author's view on this?

 

10. Do you have any data to show that M6P is colocalising with the M6P receptor?

Author Response

Reviewer 3

 

This is a very interesting and well-designed manuscript. However, a few queries need to be answered.

 

1. The data in Fig 1D are from only hBMECs. What is the expression level of different proteins in hiPSC? How do you compare the difference in expression levels?

Response:

We thank the reviewer for this important clarification request. The comparison of protein expression levels between hiPSCs and differentiated hBMECs was performed quantitatively by Western blot analysis and is presented in Fig. 1B and Fig. 1C. These experiments were used to evaluate changes in expression of BBB-associated proteins following differentiation.

In contrast, Fig. 1D was not intended for quantitative comparison of protein expression levels, but rather to illustrate the subcellular localization and junctional organization of key tight junction and adherens junction proteins in differentiated hBMECs. Specifically, this figure demonstrates the characteristic membrane localization and continuous junctional staining pattern expected for a mature BBB phenotype.

To avoid confusion, we have clarified this distinction in the revised Results section.

 

1. The same question applied to the data presented in Supplementary Figure 1.

 Response:

We thank the reviewer for this comment. Similar to Fig. 1D, Supplementary Figure 1 was not intended to provide a quantitative comparison of protein expression levels between hiPSCs and differentiated hBMECs. Rather, the immunofluorescence images were included to demonstrate that GLUT1 and CI-M6PR are expressed in our differentiated hiPSC-derived BBB model and display the expected cellular localization. We have clarified in the revised manuscript that Supplementary Figure 1 serves as a qualitative confirmation of protein expression and localization within the differentiated hBMEC system.

1. Please label Fig 1F more clearly, possibly with a different colour code.

Response:

We thank the reviewer for this suggestion. Figure 1F has been revised to improve clarity and readability. Specifically, the labeling and contrast have been enhanced using a clearer black-and-white scheme to facilitate interpretation of the different conditions and improve figure accessibility.

1. The idea of using M6P in conjugation is excellent. Is it possible that D-mannose or M1P conjugation will show a similar effect?

Response:

We thank the reviewer for this interesting question. Based on the current understanding of CI-M6PR biology and our previous work, we do not expect D-mannose or mannose-1-phosphate (M1P) conjugation to reproduce the same effect observed with M6P-functionalized liposomes.

The cation-independent mannose-6-phosphate receptor recognizes the specific spatial arrangement of the phosphate group at the C6 position of mannose, which is critical for high-affinity ligand binding. D-mannose lacks this phosphate group entirely, while M1P contains phosphorylation at a different position that is not known to efficiently interact with CI-M6PR binding domains. In our previous and current study (Ref. 25), structurally related glycans and similarly charged control ligands did not reproduce the enhanced uptake observed with M6P-functionalized liposomes, as well as just competition with free M6P inhibited the upted, supporting the specificity of the M6P effect beyond simple carbohydrate decoration or surface charge.

Nevertheless, we agree that systematic comparison of alternative glycan ligands could provide valuable insight into receptor selectivity and glycan-mediated BBB transport mechanisms.

 

1. From Fig 2A, it is unclear what the structural features of the liposomes are. What is the composition in the core and in the outer layer? Show the structure of PEG-conjugated M6P.

Response:

We thank the reviewer for this helpful comment. We agree that additional clarification regarding the structural organization of the liposomes improves the readability of the manuscript.

The liposomes were prepared from DOPC, cholesterol, and PEG-lipid conjugates, including M6P-PEG-lipid or control PEG-lipid derivatives, which were incorporated directly into the initial lipid mixture prior to vesicle formation. As a result, the ligand-PEG-lipid conjugates are expected to distribute throughout the lipid bilayer during self-assembly, without a preferential localization to either the inner or outer leaflet. The PEG chains extend from the liposomal surface, exposing the M6P ligand to the extracellular environment for interaction with cellular uptake pathways.

To address the reviewer’s concern, we have revised Fig. 2A and the corresponding text to better describe the liposome architecture and surface organization. In addition, the chemical structures of the synthesized PEG-conjugated ligands are added as supplementary figure 2 in the revised version of the manuscript.

 

1. The data in Fig 3A do not show a significant increase in Fluorescence. Is the fluorescent marker stable inside the cells? What is the uptake efficiency of the naked FL dye?

Response:

We thank the reviewer for this comment. Figure 3A was intended primarily as a representative qualitative image showing the intracellular distribution of liposomes, whereas the quantitative comparison of uptake is provided in Figure 3B. In Figure 3B, image-based analysis demonstrated a significant increase in the number of intracellular M6P-liposome puncta compared with control liposomes after 4 h of treatment.

Regarding fluorophore stability, the liposomes were labeled with highly lipophilic membrane dyes, DiO or DiD, which preferentially partition into lipid bilayers and are commonly used to track lipid nanoparticles and liposomes. The comparable initial fluorescence intensity of control and M6P-liposome formulations was verified before biological experiments, as shown in Figure 2E. In addition, our NTA analysis after transcytosis showed that fluorescence was associated with intact particles and was abolished after Triton X-100 treatment, supporting that the detected signal mainly originated from membrane-associated fluorophore rather than free dye.

We did not include naked fluorescent dye as a separate uptake control in this study, because DiO/DiD are hydrophobic membrane tracers and do not behave as freely soluble fluorescent cargos under aqueous biological conditions. However, to avoid overinterpretation, we have clarified this point in the revised manuscript and now state that fluorescence-based uptake was used as a relative readout of liposome-associated signal, supported by formulation-matched fluorescence normalization and intact particle validation.

 

1. The values of FL intensity (Fig 3E) may provide a better visibility if provided in a table with raw values.

Response:

We thank the reviewer for this helpful suggestion. In response, we have added a supplementary table containing the raw fluorescence intensity values corresponding to Fig. 3E, together with the quantitative values from the other datasets presented in the manuscript. To improve readability and avoid excessive numerical detail in the main text, these raw values were removed from the Results section and are now provided in the Supplementary Material for easier reference. 

1. Similarly, some of the values presented in Fig. 4 can be put together in a table.

Response:

We thank the reviewer for this helpful suggestion. In response, we have added a supplementary table containing the raw fluorescence intensity values corresponding to Fig. 4, together with the quantitative values from the other datasets presented in the manuscript. To improve readability and avoid excessive numerical detail in the main text, these raw values were removed from the Results section and are now provided in the Supplementary Material for easier reference.

1. The data showed in Fig6D does not a significantly increase uptake. If an FL dye is used for a biomarker, the change in uptake level or the difference should be many more folds than shown here. What is the author's view on this?

Response:

We thank the reviewer for this important observation. We agree that the increase observed in Fig. 6D is more modest than the large fold-changes sometimes reported in direct cellular uptake studies. However, the uptake increase was statistically significant and reproducible across independent experiments.

We believe this result should be interpreted within the context of the experimental design and the biological complexity of BBB transcytosis assays. In this experiment, fluorescence was measured after multiple sequential processes, including liposome uptake by hiPSC-BMECs, intracellular trafficking, transcytosis across the endothelial barrier, recovery in the basolateral compartment, and subsequent uptake by downstream neuronal cells. Each of these steps can attenuate the magnitude of the final detectable signal relative to direct exposure assays. Consequently, even moderate but significant increases in downstream fluorescence may reflect biologically meaningful improvements in BBB transport efficiency.

Importantly, the increased downstream uptake correlated with the enhanced transcytosis observed in the endothelial BBB model. Furthermore, NTA analysis demonstrated recovery of intact fluorescent particles after BBB crossing, supporting that the measured fluorescence originated predominantly from transported liposomes rather than free dye alone.

 

1. Do you have any data to show that M6P is colocalising with the M6P receptor?

Response:

We thank the reviewer for this important question. At present, we do not have direct co-localization data between M6P-functionalized liposomes and CI-M6PR in the hiPSC-BMEC system. The main technical limitation is related to the immunofluorescence protocol required for detection of CI-M6PR with the antibody used in this study.

Specifically, the antibody epitope recognized in our IF experiments is located within the cytoplasmic C-terminal domain of CI-M6PR, which requires cell permeabilization with Triton X-100 to allow antibody access. However, Triton X-100 disrupts lipid membranes and solubilizes liposomes, resulting in loss of the fluorescent liposomal signal and preventing reliable visualization of intact intracellular liposome localization during co-localization analysis. We tried different conditions and we were not successful, also considering the low plasma membrane expression of CI-M6PR.

Therefore, direct co-localization experiments could not be performed under conditions that simultaneously preserve both CI-M6PR immunostaining and liposome integrity. We agree with the reviewer that obtaining such data would be highly interesting and valuable for further mechanistic characterization of the uptake pathway.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have satisfactorily responded to the comments made earlier. Therefor the manuscript may be accepte for publicaiton.