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Cited by 72RNA Interference and Gene DeliveryBarrier Structure and Function StudiesNanoparticle-Based Drug Delivery

Predictive High-Throughput Platform for Dual Screening of mRNA Lipid Nanoparticle Blood–Brain Barrier Transfection and Crossing

Open access
Han, Emily L., Padilla, Marshall S., Palanki, Rohan, Kim, Dongyoon, Mrksich, Kaitlin, Li, Jacqueline J., Tang, Sophia, Yoon, Il-Chul +1 more·2024·Nano Letters
DOI 10.1021/acs.nanolett.3c03509PMC PMC11922166
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Abstract

Lipid nanoparticle (LNP)-mediated nucleic acid therapies, including mRNA protein replacement and gene editing therapies, hold great potential in treating neurological disorders including neurodegeneration, brain cancer, and stroke. However, delivering LNPs across the blood–brain barrier (BBB) after systemic administration remains underexplored. In this work, we engineered a high-throughput screening transwell platform for the BBB (HTS-BBB), specifically optimized for screening mRNA LNPs. Unlike most transwell assays, which only assess transport across an endothelial monolayer, HTS-BBB simultaneously measures LNP transport and mRNA transfection of the endothelial cells themselves. We then use HTS-BBB to screen a library of 14 LNPs made with structurally diverse ionizable lipids and demonstrate it is predictive of in vivo performance by validating lead candidates for mRNA delivery to the mouse brain after intravenous injection. Going forward, this platform could be used to screen large libraries of brain-targeted LNPs for a range of protein replacement and gene editing applications.

AI snapshot

Written from the paper’s full text.

TL;DR

The paper develops HTS-BBB, a 96-well transwell platform that simultaneously measures mRNA lipid nanoparticle transfection of brain endothelial cells and transport across the blood–brain barrier monolayer. Screening 14 ionizable-lipid LNPs identified lead candidates whose in vitro performance predicted mRNA delivery to the mouse brain after intravenous injection.

Key findings

  • HTS-BBB was optimized with hCMEC/D3 cells seeded at 30,000 cells/cm², forming a complete monolayer by Day 6; 10 kDa FITC-dextran transport was ~5%, 70 kDa was ~2%, and TEER was ~20 Ω/cm².
  • Luciferase mRNA with 1 mol % DiR gave the most sensitive readout; luminescence outperformed mCherry fluorescence, higher DiR amounts lowered transfection, and 24 h treatment gave higher signal than 6 h.
  • Screening 14 LNPs showed statistically significant differences in transport across the endothelial monolayer, but monolayer transfection did not correlate with transport, and neither LNP size nor zeta potential correlated with transport.
  • In vivo, LNP 4 and LNP 10 gave the greatest brain signal; LNP 4 had about 1,000-fold greater signal than LNP 3, and the brain contributed approximately 1% of total organ luminescence for LNP 4 versus approximately 0.2% for LNP 3. A second study showed both LNP 3 and LNP 4 transported into the brain by DiR signal, but only LNP 4 produced significant mCherry transfection signal.

Methods

  • Built a 96-well transwell HTS-BBB with an hCMEC/D3 brain endothelial monolayer on a semipermeable membrane, separating apical blood-side and basolateral brain-side compartments; optimized seeding density, growth time, junction markers, permeability, and TEER.
  • Screened a library of 14 LNPs with structurally diverse ionizable lipids, each carrying luciferase mRNA and 1 mol % DiR, for endothelial transfection and transport; also assessed physicochemical properties and a proof-of-concept co-culture with differentiated neurons.
  • Validated lead LNPs in healthy adult C57BL/6 mice after intravenous injection: first with luciferase mRNA at 0.3 mg/kg and IVIS imaging at 6 h, then with DiR and mCherry mRNA at 0.5 mg/kg and sacrifice at 24 h.

Limitations

  • The paper states HTS-BBB is relatively simple, containing only endothelial cells plus a proof-of-concept neuron co-culture; future work may add astrocytes, pericytes, microglia, or neuroimmune components. It also notes that in vitro and in vivo kinetics likely vary.
  • Evident from the text: the platform relies on the immortalized hCMEC/D3 cell line, TEER was measured in 12-well transwells rather than the 96-well HTS-BBB due to electrode compatibility, DLS could not detect LNPs in media because of serum interference, and transport readout depends on DiR dye plus Triton X-100.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1021/acs.nanolett.3c03509

Cites

48 of 48 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

14 of 20 cited sources checked · 7 supported · 0 flagged · accuracy not yet measured

  • Findingcitation

    TEER was around 20 Ω/cm2 on Day 6 after cell seeding, consistent with other reported values.

    “The TEER was around 20 Ω/cm2 on Day 6 after cell seeding, consistent with other reported values (Figure S3).37,38”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [37] source
    • cites [38] source
  • Findingown data

    The optimized monolayer strongly expressed ZO-1 and VE-cadherin.

    “Moreover, the optimized monolayer strongly expressed ZO-1 and VE-cadherin, proteins which contribute to tight and adherens junction integrity (Figure 2C).36”
    Quote in the paperSubstantial claim
  • Findingown data

    Luminescence had a much more sensitive readout than mCherry fluorescence for assessing mRNA transfection.

    “However, after assessing the fold change in signal over untreated cells for both mRNAs in a traditional 96-well plate, luminescence had a much more sensitive readout (Figure 3D).”
    Quote in the paperSubstantial claim
  • Findingown data

    Higher amounts of DiR may lower LNP transfection ability.

    “Interestingly, there was also an effect from the amount of DiR added, indicating that higher amounts of DiR may lower LNP transfection ability.”
    Quote in the paperNot a substantial claim
  • Findingown data

    A complete monolayer without overgrowth formed at Day 6 after initial cell seeding with an initial seeding density of 30,000 cells/cm2.

    “A complete monolayer without overgrowth was formed at Day 6 after initial cell seeding with an initial seeding density of 30,000 cells/cm2 (Figure 2B).”
    Quote in the paperSubstantial claim
  • May combine two claims. 21
  • Classified as probably not a substantial claim. 5

  • May combine two claims.

    Premisecitation

    BBB transwell models allow transport measurements but are low-throughput and do not capture transfection.

    “BBB transwell models have a semipermeable membrane separating two compartments allowing for transport measurements but are low-throughput as they are typically only used in 12- or 24-well formats and do not capture transfection.30,31”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [30] source

      The Monolayer Models section says transwell chambers have a semipermeable membrane separating two sides and allowing solute exchange, supporting transport measurements. It describes transwell models as scalable rather than low-throughput, and our copy does not support the claimed 12- or 24-well restriction or say that they cannot capture transfection (page numbers unavailable). (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [31] source

      The Introduction says transwell models provide access to apical and basolateral compartments for drug application and sampling, supporting the transport-measurement point. The Methods compare 12- and 24-well setups, but do not establish that transwell models are typically limited to those formats or are low-throughput. No passage found addresses transfection. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    The blood-brain barrier prevents approximately 98% of small molecule drugs and approximately 100% of large molecule drugs from entering the brain.

    “prevents approximately 98% of small molecule drugs and approximately 100% of large molecule drugs from entering the brain.28”
    Quote in the paperSubstantial claim1 of 1 sources checked

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The highly modular nature of LNPs allows them to be optimized for organ- and cell type-specific delivery following systemic administration.

    “Furthermore, the highly modular nature of LNPs allows them to be optimized for organ- and cell type-specific delivery following systemic administration.23–25”
    Quote in the paperNot a substantial claim2 of 3 sources checked
    • cites [23] source
    • cites [24] source
    • cites [25] source

  • May combine two claims.

    Premisecitation

    Brain endothelial monolayers grown in traditional 96-well plates can screen for transfection in a high-throughput manner but cannot measure transport across the blood-brain barrier.

    “Brain endothelial monolayers grown in traditional 96-well plates can screen for transfection in a high-throughput manner, but the lack of two compartments means that they cannot measure transport across the BBB.30”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [30] source

      The Monolayer Models section says monocultures in Petri dishes lack separate blood and brain compartments and are unsuitable for studying drug transport across the BBB. It does not identify traditional 96-well plates or say these cultures can screen for transfection in a high-throughput manner. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    hCMEC/D3 cells form contact-inhibited monolayers when grown on collagen type 1 and express tight and adherens junctions.

    “Immortalized human microvascular brain endothelial (hCMEC/D3) cells were chosen as the cell line as they (i) are able to form contact-inhibited monolayers when grown on collagen type 1, a key component of the BBB basement membrane, and (ii) express tight and adherens junctions, which help regulate the structural integrity and permeability of the BBB.35,36”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [35] source
    • cites [36] source

      The section on BBB dysregulation identifies hCMEC/D3 as a human brain endothelial cell line and reports loss of tight-junction proteins after barrier disruption. The section on VE-cadherin describes adherens and tight junctions in brain endothelial cells generally, but does not establish both for hCMEC/D3 specifically. Our copy does not report contact-inhibited growth on collagen type I; its basement-membrane section lists collagen type IV instead. No page numbers are available in our copy. (LLM, conf=0.85)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Interpretationcitation

    These FITC-dextran transport values are consistent with other values reported in literature for in vitro BBB models and demonstrate that the monolayer possesses high structural integrity and selective permeability.

    “These values are consistent with other values reported in literature for in vitro BBB models and demonstrate that the monolayer possesses high structural integrity and selective permeability.37,38”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [37] source
    • cites [38] source

      The Discussion (page not provided) says its FITC-dextran permeability values are in range with previous work, and the Results report that both monolayers restricted FITC-dextran passage. But the paper distinguishes the tighter ihBMEC barrier from the leakier bEnd.3 barrier; it does not establish that the citing paper’s unspecified monolayer has high structural integrity and selective permeability. (LLM, conf=0.85)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    LNP 4 with ionizable lipid C12-494 was previously shown to transfect endothelial cells in the placenta after intravenous injection and induce vasodilation in the placenta after VEGF mRNA delivery.

    “LNP 4, with ionizable lipid C12–494, was previously shown to transfect endothelial cells in the placenta after intravenous injection, as well as induce vasodilation in the placenta after VEGF mRNA delivery.40”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [40] source

      The Results sections on placental-cell delivery and VEGF-mediated vasodilation report that LNP A4 delivered mRNA to placental endothelial cells in pregnant mice and increased fetal blood-vessel area after VEGF mRNA treatment. The paper describes intravenous tail-vein injection, but it identifies the formulation as LNP A4, not “LNP 4,” and does not identify its ionizable lipid as C12–494. Those formulation details are unsupported by our copy. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Pardi et al. found that after intravenous injection of luciferase mRNA LNPs, the highest measured luminescence was at 0.2 days.

    “For instance, Pardi et al. found that after intravenous injection of luciferase mRNA LNPs, the highest measured luminescence was at 0.2 days.47”
    Quote in the paperNot a substantial claim1 of 1 sources checked

  • Classified as probably not a substantial claim.

    Premisecitation

    Differences in LNP brain delivery are likely a result of chemical properties rather than physical properties.

    “This agrees with the conclusion made by Ma et al. that differences in LNP brain delivery are likely a result of chemical properties rather than physical properties.29”
    Quote in the paperSubstantial claim1 of 1 sources checked

  • May combine two claims.

    Interpretationcitation

    LNPs 11 and 12 low transfection was expected since they were designed by Ma et al. to cross the blood-brain barrier via active transport.

    “Furthermore, LNPs 11 and 12 which have neurotransmitter-derived ionizable lipids had low brain endothelial transfection, which was expected since they were designed by Ma et al. to cross the BBB via active transport.29”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [29] source

      The Introduction (page unavailable) says Ma et al. designed neurotransmitter-derived lipids for BBB delivery, drawing on evidence that some tryptamine derivatives cross by active transport. The Discussion (page unavailable) reports that NT1-lipidoid LNPs crossed the BBB, but says receptor-mediated transcytosis is only a possibility and the mechanism needs further study. The paper does not report low brain-endothelial transfection or establish that such a result was expected. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Findingown data

    All LNP formulations with luciferase or mCherry mRNA and varying molar percentages of DiR were approximately 100 nm in size, with slight differences in polydispersity and similar mRNA encapsulation efficiencies around 90%.

    “All formulations were approximately 100 nm in size, with slight differences in polydispersity and similar mRNA encapsulation efficiencies of around 90% (Figure 3B).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Adding surfactant Triton X-100 to media prior to fluorescence readings increased DiR signal, likely by disrupting LNPs.

    “First, we found that adding the surfactant Triton X-100 to the media prior to taking fluorescence readings increased DiR signal, likely by disrupting the LNPs (Figure S4).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    There was a higher signal after 24 h of treatment compared to 6 h, indicating that a longer treatment period allows for greater mRNA translation.

    “Finally, there was a higher signal after 24 h of treatment compared to 6 h, indicating that a longer treatment period allows for greater mRNA translation.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    On Day 6, 10 kDa FITC-dextran displayed transport around 5%, and 70 kDa FITC-dextran around 2% across the monolayer.

    “On Day 6 after the initial seeding of cells on the transwell membrane, the 10 kDa FD displayed low transport values of around 5%, and the 70 kDa FD displayed even lower transport at around 2%, which we expected given its higher molecular weight (Figure 2D).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In HTS-BBB, luminescent readout was much more sensitive than mCherry fluorescence readout, and amount of DiR affected luminescent signal.

    “Once again, the luminescent readout was much more sensitive than the mCherry fluorescence readout, and the amount of DiR affected the luminescent signal.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Shorter growth time and lower seeding densities resulted in an incomplete monolayer, while longer growth time and higher seeding densities resulted in overconfluence of cells.

    “In comparison, shorter growth time and lower seeding densities resulted in an incomplete monolayer, while longer growth time and higher seeding densities resulted in overconfluence of cells.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The PDI of LNPs was significantly different from the PBS group after addition of FBS, pointing toward serum interference.

    “Indeed, the PDI of LNPs was significantly different from the PBS group after the addition of FBS, pointing toward serum interference.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    DLS was able to detect LNPs up to a 20-fold dilution in PBS but unable to detect LNPs at a 20-fold dilution in media.

    “While DLS was able to detect LNPs up to a 20-fold dilution in PBS, it was unable to detect LNPs at a 20-fold dilution in media (Figure S5A).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Both LNPs 3 and 4 had greater DiR signal in the brain than the PBS group, indicating that both LNPs transported into the brain, and the difference between the two LNPs was not statistically significant.

    “Both LNPs had greater DiR signal in the brain than the PBS group, indicating that both LNPs did indeed transport into the brain, and the difference between the two LNPs was not statistically significant (Figure S14A).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: DiR fluorescence signal measures intact LNP transport rather than free DiR dye transfer.

  • May combine two claims.

    Findingown data

    LNPs 4 and 10 demonstrated the greatest signal in the brain, with LNP 4 having about 1,000-fold greater signal than the bottom performer LNP 3.

    “LNPs 4 and 10 demonstrated the greatest signal in the brain, with LNP 4 having about 1,000-fold greater signal than the bottom performer LNP 3 (Figure 5A).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Luciferase luminescence and mCherry fluorescence in cells report functional mRNA transfection.
    • Rests on an assumption: Mouse brain luminescent signal after intravenous LNP injection reflects delivery and transfection in brain parenchyma rather than signal from blood or adjacent tissues.

  • May combine two claims.

    Findingown data

    LNP 4 had significantly greater mCherry signal than the PBS group, whereas LNP 3 did not.

    “LNP 4 had significantly greater mCherry signal than the PBS group, whereas LNP 3 did not (Figure S14B), which agreed with our first in vivo study using luciferase mRNA.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Luciferase luminescence and mCherry fluorescence in cells report functional mRNA transfection.

  • May combine two claims.

    Findingown data

    For LNP 4, the brain contributed approximately 1% of the signal, whereas for LNP 3 the brain only contributed approximately 0.2%.

    “We then compared the luminescent signal that each organ contributed to the total organ luminescence and found that for LNP 4, the brain contributed approximately 1% of the signal, whereas for LNP 3 the brain only contributed approximately 0.2% (Figure 5B).”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: Mouse brain luminescent signal after intravenous LNP injection reflects delivery and transfection in brain parenchyma rather than signal from blood or adjacent tissues.

  • May combine two claims.

    Interpretationreasoning

    Screening large libraries of LNPs on a single plate rather than across multiple 12- or 24-well transwell plates achieves more consistent results, saves material costs, and increases workflow speed.

    “by being able to screen large libraries of LNPs on a single plate rather than across multiple 12- or 24-well transwell plates, we can achieve more consistent results, save material costs by requiring less cells and media, and increase workflow speed by using multichannel pipettes and potentially automated robotic handling in the future.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S2 (checkable).

    Premisecitation

    Ionizable lipid nanoparticles are an ideal delivery vehicle for nucleic acid therapeutics.

    “Ionizable lipid nanoparticles (LNPs) are an ideal delivery vehicle for nucleic acid therapeutics.19”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [19] source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationreasoning

    In vitro and in vivo kinetics likely vary due to the presence of additional physical forces and biological phenomena present in vivo.

    “It is worth noting that in vitro and in vivo kinetics likely vary due to the presence of additional physical forces and biological phenomena present in vivo.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    LNPs found to have high transfection and LNPs found to have high transport could both be used to treat neurological disorders.

    “Importantly, LNPs found to have high transfection and LNPs found to have high transport could both be used to treat neurological disorders.”
    Quote in the paperSubstantial claim
26 of 26

  • The paper relies on this without support.

    DiR fluorescence signal measures intact LNP transport rather than free DiR dye transfer.

    “LNPs were formulated with luciferase or mCherry mRNA and varying molar percentages of DiR, a fluorescent lipophilic dye for tracking transport across endothelial monolayers.”
    “Both LNPs had greater DiR signal in the brain than the PBS group, indicating that both LNPs did indeed transport into the brain”

  • The paper relies on this without support.

    Luciferase luminescence and mCherry fluorescence in cells report functional mRNA transfection.

    “LNPs were formulated with luciferase or mCherry mRNA and varying molar percentages of DiR, a fluorescent lipophilic dye for tracking transport across endothelial monolayers.”
    “LNP 4 had significantly greater mCherry signal than the PBS group, whereas LNP 3 did not”

  • The paper relies on this without support.

    Mouse brain luminescent signal after intravenous LNP injection reflects delivery and transfection in brain parenchyma rather than signal from blood or adjacent tissues.

    “Mice were then subjected to whole-body imaging to capture in vivo luminescent signal in the brain using an in vivo imaging system (IVIS) (Figure S12) or euthanized for organ collection, IVIS imaging, and quantification (Figure S13).”
    “LNPs 4 and 10 demonstrated the greatest signal in the brain”
3 of 3

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Authors
Han, E. L., Padilla, M. S., Palanki, R., Kim, D., Mrksich, K., Li, J. J., Tang, S., Yoon, I.-C., & Mitchell, M. J.
Affiliations
  • University of Pennsylvania (US)
  • Children's Hospital of Philadelphia (US)
  • Abramson Cancer Center (US)
Corresponding author
Mitchell, Michael J.
ORCID
Venue
Nano Letters
Publisher
American Chemical Society (ACS)
Published
2024-01-23
Volume
24
Issue
5
Pages
1477-1486
ISSN
  • 1530-6984 (ISSN-L)
  • 1530-6992
DOI
10.1021/acs.nanolett.3c03509
PMC
PMC11922166
OpenAlex
W4391135116
Semantic Scholar
c0b6b779977db7fc613d1761f0b2d39497418658
Cited by
72
References
48
Citations by year
2026: 18 · 2025: 37 · 2024: 17
FWCI
8.32
Type
journal article
Language
en
Open access
Licence
Locations
  • Nano Letters (publishedVersion)
  • PubMed (publishedVersion)
  • PubMed Central (submittedVersion)
Funders
  • National Center for Advancing Translational Sciences (DP2 TR002776)
  • National Institute of Dental and Craniofacial Research (T90DE030854)
  • Division of Graduate Education (1845298)
  • NHLBI Division of Intramural Research (F30HL16246501A1)
  • American Cancer Society (RSG-22-122-01-ET)
  • Division of Chemical, Bioengineering, Environmental, and Transport Systems (CBET-2145491)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
  • Life Sciences › Neuroscience › Neurology › Barrier Structure and Function Studies
  • Physical Sciences › Materials Science › Biomaterials › Nanoparticle-Based Drug Delivery
Keywords
blood-brain barrier, mRNA delivery, high-throughput screening, transwell assay, ionizable lipids, endothelial cells, brain drug delivery, intravenous administration, neurodegeneration, genome editing, protein replacement therapy, predictive screening
MeSH terms
Animals, Blood-Brain Barrier, Lipids, Liposomes, RNA, Messenger, Transfection, RNA, Small Interfering, Endothelial Cells, Mice, Nanoparticles

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