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Cited by 165RNA Interference and Gene DeliveryNanoparticle-Based Drug DeliveryBarrier Structure and Function Studies

Lipid Nanoparticle Delivery of siRNA to Silence Neuronal Gene Expression in the Brain

Open access
Rungta, Ravi L, Choi, Hyun B, Lin, Paulo JC, Ko, Rebecca WY, Ashby, Donovan, Nair, Jay, Manoharan, Muthiah, Cullis, Pieter R +1 more·2013·Molecular Therapy—Nucleic Acids
DOI 10.1038/mtna.2013.65PMC PMC3889191
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Abstract

Manipulation of gene expression in the brain is fundamental for understanding the function of proteins involved in neuronal processes. In this article, we show a method for using small interfering RNA (siRNA) in lipid nanoparticles (LNPs) to efficiently silence neuronal gene expression in cell culture and in the brain in vivo through intracranial injection. We show that neurons accumulate these LNPs in an apolipoprotein E-dependent fashion, resulting in very efficient uptake in cell culture (100%) with little apparent toxicity. In vivo, intracortical or intracerebroventricular (ICV) siRNA-LNP injections resulted in knockdown of target genes either in discrete regions around the injection site or in more widespread areas following ICV injections with no apparent toxicity or immune reactions from the LNPs. Effective targeted knockdown was demonstrated by showing that intracortical delivery of siRNA against GRIN1 (encoding GluN1 subunit of the NMDA receptor (NMDAR)) selectively reduced synaptic NMDAR currents in vivo as compared with synaptic AMPA receptor currents. Therefore, LNP delivery of siRNA rapidly manipulates expression of proteins involved in neuronal processes in vivo, possibly enabling the development of gene therapies for neurological disorders.Molecular Therapy-Nucleic Acids (2013) 2, e136; doi:10.1038/mtna.2013.65; published online 3 December 2013.

AI snapshot

Written from the paper’s full text.

TL;DR

Lipid nanoparticles (LNPs) deliver siRNA efficiently to neurons in culture and in the brain after intracranial or intracerebroventricular injection, silencing target genes with little apparent toxicity. Uptake is apolipoprotein E–dependent, and GRIN1 knockdown functionally reduces synaptic NMDA receptor currents in vivo.

Key findings

  • In cultured hippocampal neurons, 100% of neurons took up DiI-labeled LNPs after 24 hours; PTEN siRNA (246 nmol/l, 48 hours) reduced PTEN/β-actin by 80% versus control (P < 0.001), and 0.7 nmol/l still reduced PTEN/β-actin by 59% (P < 0.001), with no toxicity by LDH release or nuclear morphology.
  • Intracortical LNP-PTEN siRNA reduced PTEN/β-actin by 72% versus noninjected and 69% versus luc siRNA–injected controls (P < 0.001); knockdown was significant within 1 mm of the injection tract but not 1–3 mm away, and was sustained for at least 15 days (91% reduction, P < 0.001).
  • Bilateral intracerebroventricular injection of 2 µl PTEN siRNA-LNPs produced widespread uptake and reduced PTEN/β-actin by 55.8% in hippocampus and 51.2% in striatum versus control siRNA–injected rats.
  • GRIN1 siRNA reduced GluN1/β-actin by 51% versus noninjected and 54% versus luc siRNA-LNP controls (P < 0.01 and P < 0.05), decreased GluN1 cluster density in vitro by 41% (P < 0.001), and selectively reduced the synaptic NMDAR/AMPAR current ratio in vivo; TNF-α was not significantly increased by LNP-siRNA in slices or injected tissue (P > 0.05).

Methods

  • LNPs were prepared by microfluidic mixing with DMAP-BLP/DSPC/cholesterol/PEG-DMG at 50/10/37.5/1.5 mol% plus 1 mol% DiOC18 or DiIC18; PTEN siRNA-LNPs had diameter 55 ± 11 nm, and in vivo LNPs were 50–60 nm.
  • Primary rat hippocampal neuron cultures (DIV 12–14) with an astrocyte feeder layer were used for uptake, toxicity, and knockdown assays; Sprague–Dawley rats (P22–P26) received intracortical injections (500 nl at 5 mg/ml siRNA) or bilateral ICV injections (2 µl), with brain slices prepared about 5 days later.
  • Main analyses included Western blot, immunohistochemistry/immunocytochemistry, LDH assay, DiI uptake assay, TNF-α ELISA, and whole-cell patch-clamp measurement of NMDAR/AMPAR excitatory postsynaptic currents; statistics used t-test or ANOVA with Newman–Keuls multiple comparison test.

Limitations

  • The paper states that no further optimization was done to increase LNP-siRNA efficiency in this study, and that the therapeutic utility of direct intracranial LNP-siRNA administration for neurological disorders remains to be established.
  • The paper notes that LNP systems cannot cross the blood–brain barrier, that local intracortical knockdown was limited to about 1 mm from the injection tract, and that the longest time point tested for PTEN suppression was 15 days.
  • Evident from the text: findings are limited to primary rat neuronal cultures and rats, with no primate or human data; the in vivo functional experiments also contain a likely typo referring to “GluR1 siRNA” where GluN1/GRIN1 siRNA was intended.

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

Integrity signals

Registry

Crossref · 10.1038/mtna.2013.65

Cites

36 of 36 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

17 of 34 cited sources checked · 7 supported · 3 flagged · accuracy not yet measured

  • Premisecitation

    Current methods to deliver siRNA to cultured neurons (electroporation, calcium phosphate, or lipofection) usually result in transfection rates of 1-10%.

    “which usually result in transfection rates of 1–10%.13”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [13] source

      The calcium-phosphate table entry gives typical efficiencies of 5–10% in postmitotic neurons, and the Lipofection section gives 1–5% for plasmid-based transfection. Those figures do not establish the claimed usual rate for siRNA delivery: the Lipofection section instead describes efficient siRNA transfer, reaching up to 83% in primary rat hippocampal neurons. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
  • Findingown data

    LNP uptake reached saturation at ~5 μg ApoE/ml.

    “We found that LNP uptake reached saturation at ˜5 µg ApoE/ml (Figure 3c), in line with measurements from human cerebrospinal fluid (CSF) in vivo of 9.09 µg/ml.16”
    Quote in the paperSubstantial claim1 of 1 sources checked
  • Premisecitation

    In the brain and neuronal cultures, ApoE is synthesized in astrocytes.

    “In the brain and neuronal cultures, ApoE is synthesized in astrocytes.14,15”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [14] source
    • cites [15] source

      The Biochemical properties section states that astrocytes are the predominant source of ApoE in the brain and describes ApoE particles secreted by cultured astrocytes. It does not specifically establish where ApoE is synthesized in neuronal cultures, and it notes that microglia also synthesize some ApoE. Page numbers are unavailable in our copy. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
  • Premisecitation

    Viral delivery is the most effective available way to transfect neurons.

    “Viral delivery is the most effective available way to transfect neurons.13,24”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [13] source

      The “Virus-based transfection methods” section describes viral delivery as extremely efficient and often the only viable option for efficient delivery to specific cell types in vivo. It does not call viral delivery the most effective method for neurons generally; the “Electrical transfection methods” section instead calls nucleofection the method of choice when high transfection efficiency is essential. (LLM, conf=0.85)

      Experimental — accuracy not yet measured
    • cites [24] source
  • Premisecitation

    Expression of GluN1 is necessary for the formation of a functional NMDAR channel in the plasma membrane.

    “Expression of GluN1 is necessary for the formation of a functional NMDAR channel in the plasma membrane.19”
    Quote in the paperSubstantial claim1 of 1 sources checked
  • May combine two claims. 21
  • Classified as probably not a substantial claim. 20

  • May combine two claims.

    Interpretationcitation

    LNPs are much more efficient and less toxic than current methods used to deliver siRNA to cultured neurons such as electroporation, calcium phosphate, or lipofection.

    “These results indicate that LNPs are much more efficient and less toxic than current methods used to deliver siRNA to cultured neurons such as electroporation, calcium phosphate, or lipofection, which usually result in transfection rates of 1–10%.13”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [13] source

      The Lipofection section reports siRNA delivery into postmitotic neurons at up to 83% efficiency, contrary to the cited sentence’s characterization of siRNA-delivery methods as usually achieving 1–10%. Its low lipofection rates of typically 1–5% are explicitly for plasmid-based vectors, not siRNA. The paper does not compare LNP efficiency or toxicity with these methods. (LLM, conf=0.88)

      Checked by a reasoning model — may be wrong.

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Current nonviral vectors exhibit transfection efficiencies of 10% or less and are often accompanied by cell damage.

    “By contrast, current nonviral vectors exhibit transfection efficiencies of 10% or less and are often accompanied by cell damage.13”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [13] source

      The Electrical transfection methods section directly contradicts the claim’s 10% ceiling: it reports nucleofection rates averaging 60–80% after optimization, with up to 95% in embryonic rat neurons and better cell survival than conventional electroporation. The paper does report cell damage with some nonviral techniques, including biolistics, but does not characterize nonviral methods as uniformly low-efficiency and damaging. Page numbers were not available in our copy. (LLM, conf=0.90)

      Checked by a reasoning model — may be wrong.

      Experimental — accuracy not yet measured

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

    Premisecitation

    A number of clinical trials are now in progress for treatments of serious neurological diseases that rely on continuous infusion of ASOs into the CSF.

    “Furthermore, a number of clinical trials are now in progress for treatments of serious neurological diseases that rely on continuous infusion of ASOs into the CSF.27,28”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [27] source
    • cites [28] source

      The Discussion (page unavailable) describes a Phase I trial in human familial ALS patients as planned, not in progress, and says safety studies were underway in animals. The Results describe continuous ASO infusion in rats and a nonhuman primate, but the paper does not report multiple ongoing clinical trials. (LLM, conf=0.88)

      Checked by a reasoning model — may be wrong.

      Experimental — accuracy not yet measured

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

    Premisecitation

    LNP systems with this lipid composition can silence target genes in hepatocytes following IV injection at dose levels as low as 0.01 mg siRNA/kg body weight in an apolipoprotein E (ApoE)-dependent fashion.

    “Previous work has shown that LNP systems with this lipid composition can silence target genes in hepatocytes following IV injection at dose levels as low as 0.01 mg siRNA/kg body weight8,9 in an apolipoprotein E (ApoE)–dependent fashion.10”
    Quote in the paperNot a substantial claim2 of 3 sources checked
    • cites [8] source
    • cites [9] source
    • cites [10] source
    • Rests on an assumption: ApoE is the dominant lipoprotein in the brain.

  • Classified as probably not a substantial claim.

    Premisecitation

    The NMDAR ion channel is heteromeric, composed of two obligatory GluN1 subunits and two GluN2 or GluN3 subunits.

    “The NMDAR ion channel is heteromeric, composed of two obligatory GluN1 subunits and two GluN2 or GluN3 subunits.”
    Quote in the paperSubstantial claim1 of 1 sources checked

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

    Premisecitation

    Injection of ASOs into the CSF represents another promising method to silence neuronal gene expression in vivo.

    “Injection of ASOs into the CSF represents another promising method to silence neuronal gene expression in vivo.25”
    Quote in the paperNot a substantial claim1 of 1 sources checked

  • May combine two claims.

    Premisecitation

    The LNP delivery systems used here contain the ionizable cationic lipid DMAP-BLP, a lipid that exhibits optimized bilayer destabilizing and pKa properties leading to highly potent gene silencing in hepatocytes following IV administration similar to "gold standard" lipids such as DLinMC3-DMA.

    “With respect to the mechanism of action, the LNP delivery systems used here contain the ionizable cationic lipid DMAP-BLP, a lipid that exhibits optimized bilayer destabilizing and pKa properties leading to highly potent gene silencing in hepatocytes following IV administration (Supplementary Data) that is similar to “gold standard” lipids such as DLinMC3-DMA.8”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [8] source

      Split into 2 atoms — 0 confirmed, 1 partially supported, 0 disputed, 0 misattributed, 1 unverifiable. Expand the claim to see per-atom verdicts.

      Experimental — accuracy not yet measured

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

    Premisecitation

    There are now well-established methods to reduce or eliminate siRNA immunogenicity by adjusting siRNA chemistry.

    “In addition, there are now well-established methods to reduce or eliminate siRNA immunogenicity by adjusting siRNA chemistry.29”
    Quote in the paperNot a substantial claim1 of 1 sources checked

  • May combine two claims.

    Premisecitation

    Two major problems associated with ASOs are the high doses required to achieve gene silencing effects and immunostimulatory effects.

    “However, two major problems associated with ASOs are the high doses required to achieve gene silencing effects and immunostimulatory effects.26,27”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [26] source

      The Cellular Delivery and Associated Safety section says phosphorothioate antisense oligonucleotides (AONs) can have immunostimulatory effects, while noting that appropriate modifications can suppress them. Neither that section nor the AON Mechanisms section says high doses are required to achieve gene silencing; the paper instead discusses dosage control and possible dose-dependent toxicity. Our copy provides no page numbers for these passages. (LLM, conf=0.87)

      Experimental — accuracy not yet measured
    • cites [27] source

  • May combine two claims.

    Findingown data

    In vivo siRNA-LNP injections caused no apparent toxicity or immune reactions from the LNPs.

    “In vivo, intracortical or intracerebroventricular (ICV) siRNA-LNP injections resulted in knockdown of target genes either in discrete regions around the injection site or in more widespread areas following ICV injections with no apparent toxicity or immune reactions from the LNPs.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    LNPs were nontoxic at the concentrations used in cell culture, with no observable differences in cell death relative to controls.

    “LNPs were nontoxic at the concentrations used, with no observable differences in cell death relative to controls measured either by comparing nuclear morphology or by lactate dehydrogenase (LDH) release (Figure 2c).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    LNP-siRNA can efficiently silence neuronal gene expression in cell culture and in the brain in vivo through intracranial injection.

    “In this article, we show a method for using small interfering RNA (siRNA) in lipid nanoparticles (LNPs) to efficiently silence neuronal gene expression in cell culture and in the brain in vivo through intracranial injection.”
    • Also stated in: § Introduction · boosted
      “In this article, we report the conditions under which LNP delivery of siRNA is a remarkably efficient method for silencing neuronal gene expression in both primary neuronal culture and following intracranial injection in vivo.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    With no added ApoE, there was no evidence of neuronal LNP uptake, whereas graded uptake was observed when ApoE concentrations were increased above 0.1 μg/ml to 1 μg/ml and 5 μg/ml.

    “With no added ApoE, there was no evidence of neuronal LNP uptake, whereas graded uptake was observed when ApoE concentrations were increased above 0.1μg/ml to 1 μg/ml and 5 µg/ml ApoE.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    PTEN protein levels were significantly reduced at distances within 1 mm of the injection tract but not 1-3 mm away.

    “Western blot analysis, which revealed that PTEN protein levels were significantly reduced at distances within 1 mm of the injection tract but not 1–3 mm away (Figure 5b).”
    Quote in the paperSubstantial claim

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

    Findingown data

    Positive control 1% Triton X-100 showed a large increase in LDH release (90.21 ± 0.62%).

    “As a positive control, cultures treated with 1% Triton X-100 showed a large increase in LDH release (90.21 ± 0.62%).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    A single LNP-siRNA injection of 2.5 μg of siRNA per rat by intracortical administration results in pronounced (up to 91%) gene silencing that is sustained for at least 15 days after injection.

    “In this article, we demonstrate that a single LNP-siRNA injection of 2.5 µg of siRNA per rat by intracortical administration results in pronounced (up to 91%) gene silencing that is sustained for at least 15 days after injection.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In experiments in vivo, localized injection of siRNA-LNPs within the cortex or ICV injections led to consistent decreases in both mRNA and protein expression with no indication of cell loss or damage from the uptake of LNPs.

    “In experiments in vivo, localized injection of siRNA-LNPs within the cortex or ICV injections to cause more diffuse and widespread LNP uptake led to consistent decreases in both mRNA and protein expression again with no indication of cell loss or damage from the uptake of LNPs.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Sequence number 1 resulted in the most robust knockdown of GluN1 and was used for subsequent in vivo experiments.

    “however, sequence number 1 resulted in the most robust knockdown, and it was therefore used for the subsequent experiments in vivo.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    There was no immunostimulatory effect when luc siRNA-LNPs (246 nmol/l) were applied to brain slices; TNF-α levels in the treated group were not significantly different from the control group (P > 0.05).

    “The results suggest that there was no immunostimulatory effect when luc siRNA-LNPs (246 nmol/l) were applied to brain slices. TNF-α levels in the luc siRNA-LNP–treated group were not significantly different from the control group (Figure 4f, P > 0.05).”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Study design and conductown data

    The NMDA component was measured as the amplitude of the outward current at Vh + 40 mV 50 ms after the stimulation artifact, a time point when the AMPAR current had returned to baseline, verified by subtracting the trace in the presence of APV.

    “To get the NMDA component, we measured the amplitude of the outward current at Vh + 40 mV 50 ms after the stimulation artifact, a time point when the AMPAR current had returned to baseline (this was verified by subtracting the trace in the presence of the NMDAR antagonist, DL-2-amino-5-phosphonopentanoic acid (APV) to show the outward INMDAR (blue) and IAMPAR (red) components separately).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    In ApoE knockout mice, LNP-siRNA gene silencing activity is inhibited in hepatocytes and can be reestablished by incubating the LNPs with ApoE before IV administration.

    “In ApoE knockout mice, LNP-siRNA gene silencing activity is inhibited in hepatocytes and can be reestablished by incubating the LNPs with ApoE before IV administration.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [10] source

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

    Premisecitation

    NMDARs are typically arranged in clusters at synaptic and extrasynaptic sites.

    “NMDARs are typically arranged in clusters at synaptic and extrasynaptic sites.20,21”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [20] source
    • cites [21] source

  • Classified as probably not a substantial claim.

    Study design and conductown data

    The AMPA component was measured as the inward current when the cell was held at −70 mV.

    “The AMPA component was measured as the inward current when the cell was held at −70 mV (Figure 8d).”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    A common protocol to test the NMDAR/AMPAR ratio in response to the synaptic release of glutamate is to measure the ratio of the evoked excitatory postsynaptic current when the NMDAR is blocked by holding the cell at −70 mV to obtain a pure AMPAR component versus when the cell is held at +40 mV to get a mixed AMPAR + NMDAR component.

    “Therefore, a common protocol to test the NMDAR/AMPAR ratio in response to the synaptic release of glutamate is to measure the ratio of the evoked excitatory postsynaptic current when the NMDAR is blocked by holding the cell at −70 mV to obtain a pure AMPAR component versus when the cell is held at +40 mV to get a mixed AMPAR + NMDAR component (Figure 8d).22,23”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [22] Sah P, 1991, J Physiol (Lond) source
    • cites [23] source
    • Rests on an assumption: Glutamate is the major excitatory neurotransmitter in the brain and acts on both AMPAR and NMDAR at synapses.

  • May combine two claims.

    Interpretationown data

    When administered directly into the brain, LNPs are capable of diffusing through the extracellular milieu to deliver siRNA to neurons and induce gene silencing at sites distant from the site of injection.

    “These results demonstrate that when administered directly into the brain, LNPs are capable of diffusing through the extracellular milieu to deliver siRNA to neurons and induce gene silencing at sites distant from the site of injection.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Study design and conductown data

    Neurons chosen for experiments were within 500 μm of the injection site and were verified to have taken up LNPs based on punctate intracellular DiI fluorescence.

    “Neurons that were chosen for experiments were within 500 µm of the injection site and were verified to have taken up LNPs based on punctate intracellular DiI fluorescence (Figure 8c), and the ratio of NMDAR/AMPAR was measured.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    LNPs can circumvent the issues of viral delivery and provide a method to rapidly test various siRNA constructs against the expression of different target proteins, and then examine the protein's function.

    “The use of LNPs can circumvent these issues, and provides a method to rapidly test various siRNA constructs against the expression of different target proteins, and then examine the protein's function.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The disadvantage of viral delivery is the requirement for constructing virus vectors that take added time, potentially cause immune responses, and raise safety concerns.

  • May combine two claims.

    Study design and conductown data

    The region 200-500 μm from the injection tract had robust LNP uptake and gene knockdown but was not damaged from the injection needle itself.

    “This is a region with robust LNP uptake and gene knockdown but not damaged from the injection needle itself, suggesting that LNPs themselves are nontoxic in vivo.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    LNP-siRNA systems can exhibit efficient gene silencing properties in neurons both in vitro and in vivo that lead to functional consequences without inducing significant toxicity.

    “The results presented in this article show that LNP-siRNA systems can exhibit efficient gene silencing properties in neurons both in vitro and in vivo that lead to functional consequences without inducing significant toxicity.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    Clinical studies suggest that the immunostimulatory effects of LNP-siRNA injections are infrequent and readily managed.

    “Clinical studies suggest that the immunostimulatory effects of LNP-siRNA injections are infrequent and readily managed, see http://www.alnylam.com/capella/wp-content/uploads/2012/07/Alnylam-ALN-TTR02-PhaseI-Results-120716.pdf.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites http://www.alnylam.com/capella/wp-content/uploads/2012/07/Alnylam-ALN-TTR02-PhaseI-Results-120716.pdf

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

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

    Study design and conductreasoning

    Cell density measurements counted all cells including partial cells, resulting in an overestimate of cell density per mm3.

    “Cell density was measured in 10 µm z-stacks of 200 × 200 µm, and all cells including partial cells were counted, therefore resulting in an overestimate of cell density/mm3.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    The extracellular solutions were supplemented with picrotoxin and elevated MgCl2 and CaCl2 to block GABAA synaptic potentials, block epileptiform activity, and minimize polysynaptic responses.

    “The extracellular solutions were supplemented with 50 µmol/l picrotoxin (Sigma) and 8 mmol/l of each MgCl2 and CaCl2 to block GABAA synaptic potentials, block epileptiform activity, and minimize polysynaptic responses.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    Cells were allowed to dialyze for at least 15 minutes before starting recordings.

    “Cells were allowed to dialyze for at least 15 minutes before starting recordings.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Interpretationnone given

    The LNP platform is highly flexible and potentially allows delivery of other materials to neurons such as drugs, fluorescent dyes and plasmids.

    “The LNP platform is highly flexible, potentially allowing delivery of other materials to neurons such as drugs, fluorescent dyes and plasmids.”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    A lipid-based formulation of cytarabine has gained clinical acceptance for treatment of brain cancer following direct administration into CSF.

    “A lipid-based formulation of cytarabine has gained clinical acceptance for treatment of brain cancer following direct administration into CSF.34”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [34] source

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

    Interpretationnone given

    The therapeutic utility of direct intracranial administration of LNP-siRNA for treatment of neurological disorders remains to be established.

    “The therapeutic utility of direct intracranial administration of LNP-siRNA for treatment of neurological disorders remains to be established; however, there are now a number of clinically accepted therapies relying on direct pumping of therapeutics into the CSF.32,33”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    The membrane holding potential was returned to −70 mV at the end of the experiment to verify that there was no change in the baseline.

    “Vhold was then returned to −70 mV at the end of the experiment to verify that there was no change in the baseline.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    Access resistance was continuously monitored during the electrophysiology experiments.

    “Access resistance was continuously monitored during the experiments.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    In the LDH assay, media containing 1% Triton X-100 was used as a positive control for cell death.

    “Media containing Triton X-100 (1%) was used as a positive control for cell death.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    In immunostaining, primary antibody was omitted as a negative control.

    “As a negative control experiment, primary antibody was omitted during the immunostaining.”
    Quote in the paperNot a substantial claim
40 of 40

  • The paper relies on this without support.

    ApoE is the dominant lipoprotein in the brain.

    “Because ApoE is the dominant lipoprotein in the brain, we tested whether ApoE facilitates uptake of LNPs by neurons in cell culture.”

  • The paper relies on this without support.

    LNP systems cannot cross the blood–brain barrier.

    “Due to the inability of LNP systems to cross the blood–brain barrier, the potency of these systems for silencing genes in brain tissue has not been investigated.”

  • The paper relies on this without support.

    Luciferase siRNA is not found in the mammalian genome.

    “Of note, in control experiments, incubation of cultures with LNPs containing siRNA against luciferase (luc), which is not found in the mammalian genome, had no effect on levels of PTEN protein (Figure 2d).”

  • The paper relies on this without support.

    The disadvantage of viral delivery is the requirement for constructing virus vectors that take added time, potentially cause immune responses, and raise safety concerns.

    “The disadvantage of viral delivery is the requirement for constructing virus vectors that take added time, potentially cause immune responses, and raise safety concerns.”

  • The paper relies on this without support.

    Glutamate is the major excitatory neurotransmitter in the brain and acts on both AMPAR and NMDAR at synapses.

    “Glutamate is the major excitatory neurotransmitter in the brain and acts on both AMPAR and NMDAR at synapses.”

  • The paper relies on this without support.

    Rapid dissociation of PEG-DMG from lipid nanoparticles upon dilution allows association of proteins such as ApoE.

    “The LNP formulation employed here used PEG-DMG, which is a type of PEG-lipid with short acyl chains that allow it to dissociate rapidly30 from LNPs upon dilution, allowing association of proteins such as ApoE.”

  • The paper relies on this without support.

    The phosphorothioate (P=S) linkage in the siRNA provides protection from exonuclease degradation.

    “The P=S linkage provided protection from exonuclease degradation, and the 2′-OMe modifications reduced potential for immunostimulatory activity and provided stability toward endonucleolytic degradation.”

  • The paper relies on this without support.

    The 2′-OMe modifications in the siRNA reduce potential for immunostimulatory activity and provide stability toward endonucleolytic degradation.

    “The P=S linkage provided protection from exonuclease degradation, and the 2′-OMe modifications reduced potential for immunostimulatory activity and provided stability toward endonucleolytic degradation.”
8 of 8

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • LNPs encapsulating siRNA injected into the brain after direct intracerebral or intracerebroventricular injection efficiently knock down neuronal gene expression.

    “Rungta et al. (2013) demonstrated that LNPs encapsulating siRNA have been injected into the brain after a direct intracerebral or intracerebroventricular (ICV) injection, and were able to efficiently knock down neuronal gene expression (16).”

    From Lipid Nanoparticles Deliver mRNA to the Brain after an Intracerebral Injection (2023) · cites this paper as [16]

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • DiI uptake assay has conflicting treatment durations

    Minor

    The Results describe measuring DiI fluorescence from ruptured cells 1 hour after treatment, whereas the Methods describe treating the cultures for 2 hours before washing and rupturing them for that measurement.

  • Functional experiment names different siRNA targets

    Minor

    The functional NMDAR/AMPAR experiment says rats received GluR1 siRNA, but the surrounding account attributes the injections and resulting reduction in NMDAR currents to GluN1 siRNA. These are different targets, so the identity of the siRNA used for this conclusion is inconsistent.

2 of 2

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Authors
Rungta, R. L., Choi, H. B., Lin, P. J., Ko, R. W., Ashby, D., Nair, J., Manoharan, M., Cullis, P. R., & MacVicar, B. A.
Affiliations
  • University of British Columbia (CA)
  • Alnylam Pharmaceuticals (United States) (US)
Corresponding author
MacVicar, Brian A
ORCID
Venue
Molecular Therapy—Nucleic Acids
Publisher
Elsevier BV
Published
2013-12-03
Volume
2
Issue
11
Pages
e136
ISSN
2162-2531 (ISSN-L)
DOI
10.1038/mtna.2013.65
PMC
PMC3889191
OpenAlex
W2166483989
Semantic Scholar
ef56b67d26457c62fc9e18d60b3153ef94defeb9
Cited by
165
References
36
Citations by year
2026: 6 · 2025: 15 · 2024: 18 · 2023: 16 · 2022: 16 · 2021: 10 · 2020: 12 · 2019: 8 · 2018: 12 · 2017: 8 · 2016: 16 · 2015: 16 · 2014: 12
FWCI
5.92
Type
journal article
Language
en
Open access
Locations
  • Molecular Therapy — Nucleic Acids (publishedVersion)
  • PubMed (publishedVersion)
  • Europe PMC (PubMed Central) (submittedVersion, cc-by-nc-nd)
  • PubMed Central (submittedVersion, cc-by-nc-nd)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
  • Physical Sciences › Materials Science › Biomaterials › Nanoparticle-Based Drug Delivery
  • Life Sciences › Neuroscience › Neurology › Barrier Structure and Function Studies
Keywords
lipid nanoparticles, siRNA delivery, apolipoprotein E, intracerebroventricular administration, intracortical injection, GRIN1, NMDA receptor, AMPA receptors, synaptic currents, neurotoxicity

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-20.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-20.

Cited sources are checked by gpt-6-sol.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

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