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This review reflects comments and contributions from Femi Arogundade. Review synthesized by Femi Arogundade & Jonny Coates.

The study presents a novel small molecule cocktail, cARLA, which synergistically enhances the properties of the blood-brain barrier (BBB) in various in vitro models. By modulating key signaling pathways, cARLA promotes BBB tightness, maturation, and brain endothelial identity, improving the predictive value of these models for drug development and nanoparticle transfer across the BBB. The findings highlight the potential of cARLA to advance research and drug development for the human brain by enhancing the reproducibility and effectiveness of BBB models. The study is comprehensive, offering insights into the molecular mechanisms and functional outcomes of cARLA treatment in diverse BBB models.

The study's strength lies in its multifaceted approach, investigating not only barrier integrity but also efflux pump activity, inflammatory response, Wnt/β-catenin signaling, and drug penetration. This broad exploration contributes to a comprehensive understanding of cARLA's impact on the BBB. The pharmacological inhibition experiments involving ICG-001 and XAV 939 provide valuable mechanistic insights into the Wnt/β-catenin pathway modulation, enhancing the study's depth.

Major comments:

• The study demonstrates a robust methodology, employing primary human brain-like endothelial cell co-cultures with pericytes and astrocytes to model the blood-brain barrier (BBB) and systematically investigates the impact of cARLA on various BBB-related parameters. The inclusion of multiple experimental readouts, such as barrier integrity assays, RNA sequencing, and functional assays for drug penetration, adds strength to the comprehensiveness of the study. However, it is important to note that the extrapolation of findings from this in vitro model to the in vivo human BBB should be done cautiously, and potential differences in the cellular context between the in vitro model and the native BBB need to be considered.

• The validity of the RNA sequencing data is supported by the rigorous bioinformatic analysis, including the identification of differentially expressed genes and functional enrichment analysis. The authors appropriately address multiple comparisons by calculating false discovery rates (FDR), enhancing the reliability of their findings. However, validation of key results using alternative methods, such as quantitative PCR or additional functional assays, would further strengthen the credibility of the RNA sequencing data.

• While the study provides compelling evidence for the potential of cARLA in modulating BBB properties, the authors appropriately acknowledge the need for further in vivo studies to validate the translational relevance of their findings. Additionally, discussions on the limitations of the in vitro model and potential challenges in translating these results into clinical applications would contribute to a more balanced interpretation of the study's implications.

• The study involves primary cell isolation from mice and rats, which introduces variability due to species differences. Further justification or discussion regarding the choice of species and potential implications on the study's translatability would enhance the paper's robustness.

• The study introduces an inflammatory response with TNF-α and IL-1β, yet the selection of cytokine concentrations might impact the physiological relevance. A discussion on the rationale behind the chosen concentrations and potential physiological implications would be beneficial.

Minor comments:

• Clarify whether the mouse and rat primary cells were cultured under identical conditions, as minor variations could impact the observed effects.

• Specify the rationale for using Cldn5 heterozygous mice and discuss how this genetic background may influence the BBB model, considering the importance of claudin-5 in tight junctions.

• Elaborate on the selection of ICG-001 and XAV 939, discussing their specific mechanisms and potential off-target effects.

• Clarify if the TEER and tracer permeability measurements were performed simultaneously or at different time points, and provide the rationale behind the chosen time points.

• Specify whether the observed changes in barrier integrity in response to inflammation are consistent across all experimental replicates and conditions.

• Could you provide additional information on why Cldn5 heterozygous mice were chosen for the study? Is there any specific rationale or characteristic related to these mice that influenced their selection?

• It would be helpful to elaborate on the rationale for using the bEnd.3 cell line in addition to primary cells. Why was this particular cell line chosen, and what advantages or limitations does it bring to the study?

• What criteria were considered in selecting the specific small molecule drugs for penetration studies, and how do their properties relate to their expected behavior at the BBB?

• What is the rationale for choosing the specific components (125 µM pCPT-cAMP + 17.5 µM Ro-20-1724 + 1 mM LiCl + 3 µM A83-01) and the treatment duration (24 h) for cARLA in the primary mouse BBB model?

• Regarding the penetration of poly(L-glutamic acid) nanoparticles, how did the targeting with reduced L-glutathione affect their penetration, and what are the implications for drug delivery strategies?

• In the nanoparticle uptake experiments, what controls were employed to ensure specificity and avoid nonspecific binding?

Comments on reporting:

• The paper states that MACE-seq datasets have been deposited in the Gene Expression Omnibus (GEO) repository. It would be beneficial to provide a direct link or clear instructions on how researchers can access these datasets. This ensures transparency and facilitates reproducibility.

• The text mentions that key experiments were repeated independently, which is good scientific practice. However, providing specific information on the number of replicates for each experiment and the rationale behind the chosen sample sizes would strengthen the reporting.

Suggestions for future studies:

• Investigate the dose-response relationship of cARLA treatment on barrier integrity. Testing a range of concentrations could provide insights into the optimal concentration for modulating the BBB without compromising cell viability.

• Explore the long-term effects of cARLA treatment on the BBB model. Assess barrier integrity, gene expression, and other relevant parameters at extended time points to understand the durability and stability of the observed effects.

• While the study focuses on Wnt/β-catenin signaling, consider exploring the crosstalk with other signaling pathways that are known to influence BBB function. This could provide a more comprehensive understanding of the molecular mechanisms involved.

• Elaborate on the mechanism of efflux pump modulation by cARLA. Investigate specific pump proteins involved, their regulation, and the impact on drug permeability. This could have implications for drug delivery to the brain.

• Since the study briefly mentions the inflammatory response, consider conducting a more detailed investigation into the inflammatory pathways activated by cARLA treatment. This could help understand the interplay between inflammation and barrier integrity.

• Given the use of both mouse and rat models, consider conducting comparative studies to explore potential species-specific differences in the response to cARLA treatment. This could have implications for translatability to human models.

• Combine transcriptomic data with other omics data (proteomics, metabolomics) to achieve a systems-level understanding of the changes induced by cARLA treatment. This holistic approach could reveal interconnected pathways and provide a more comprehensive view of BBB regulation.

Competing interests

The author declares that they have no competing interests.