Neurotransmitter-derived lipidoids (NT-lipidoids) for enhanced brain delivery through intravenous injection

Transient blood-brain barrier openings allow leakage of plasma proteins into the brain, leading to neurotoxicity, vascular pathology, and chronic neuropathologic changes.

“Disruption of the BBB, using techniques such as osmotic disruption, biochemical disruption, and ultrasound-mediated disruption, is effective to introduce drugs into the brain; however, these transient BBB openings also allows for the leakage of plasma proteins into the brain, leading to neurotoxicity, vascular pathology, and chronic neuropathologic changes in the brain (1, 7, 11).”

• cites [1] Pardridge W. M., Blood–brain barrier delivery. Drug Discov. Today 12, 54– 61 (2007). 17198973 sourceNot checked — no full text of the source
• cites [7] Dong X., Current strategies for brain drug delivery. Theranostics 8, 1481– 1493 (2018). 29556336 PMC5858162 sourcePartly supported

  The section on BBB disruption in pathological conditions reports that albumin enters the brain when the barrier leaks, but this example concerns disease rather than a transient drug-delivery opening. The sections on brain permeability enhancers and non-invasive techniques describe transient openings for drug delivery; they do not link plasma-protein leakage from those openings to neurotoxicity, vascular pathology, or chronic neuropathologic changes. No page numbers were provided in our copy. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [11] Villringer K., Cuesta B. E. S., Ostwaldt A.-C., Grittner U., Brunecker P., Khalil A. A., Schindler K., Eisenblätter O., Audebert H., Fiebach J. B., DCE-MRI blood–brain barrier assessment in acute ischemic stroke. Neurology 88, 433– 440 (2017). 28031392 sourceNot checked — no full text of the source

Treatment of central nervous system diseases is severely constrained by the blood-brain barrier.

“The treatment of central nervous system (CNS) diseases, such as neurodegenerative disorders, brain tumors, brain infections, and stroke, is severely constrained by the blood-brain barrier (BBB) because it prevents the transfer of most of small-molecule drugs and macromolecules (e.g., peptides, gene drugs, and protein drugs) into the brain (1–6).”

• cites [1] Pardridge W. M., Blood–brain barrier delivery. Drug Discov. Today 12, 54– 61 (2007). 17198973 sourceNot checked — no full text of the source
• cites [2] Barchet T. M., Amiji M. M., Challenges and opportunities in CNS delivery of therapeutics for neurodegenerative diseases. Expert Opin. Drug Deliv. 6, 211– 225 (2009). 19290842 sourceNot checked — no full text of the source
• cites [3] Collins P. Y., Patel V., Joestl S. S., March D., Insel T. R., Daar A. S., Bordin I. A., Costello E. J., Durkin M., Fairburn C., Glass R. I., Hall W., Huang Y., Hyman S. E., Jamison K., Kaaya S., Kapur S., Kleinman A., Ogunniyi A., Otero-Ojeda A., Poo M.-M., Ravindranath V., Sahakian B. J., Saxena S., Singer P. A., Stein D. J., Anderson W., Dhansay M. A., Ewart W., Phillips A., Shurin S., Walport M., Grand challenges in global mental health. Nature 475, 27– 30 (2011). 21734685 PMC3173804 sourceCould not verify

  The List and Next Steps sections discuss treatment priorities for mental, neurological and substance-use disorders, but do not address the blood-brain barrier. Searches for blood-brain barrier and drug delivery into the brain found no passage supporting the attributed constraint on CNS treatment. (LLM, conf=0.74)

  Experimental — accuracy not yet measured
• cites [4] Obermeier B., Daneman R., Ransohoff R. M., Development, maintenance and disruption of the blood-brain barrier. Nat. Med. 19, 1584– 1596 (2013). 24309662 PMC4080800 sourceSupported
• cites [5] Khorkova O., Wahlestedt C., Oligonucleotide therapies for disorders of the nervous system. Nat. Biotechnol. 35, 249– 263 (2017). 28244991 PMC6043900 sourcePartly supported

  The paper’s introductory passage says that the BBB is a substantial obstacle to treating CNS disorders with oligonucleotides: fewer than 1% of systemically delivered oligonucleotides reach the brain. Its assisted-delivery discussion also mentions small molecules with poor BBB permeability, peptides, and proteins. It does not establish the citing sentence’s broader assertion that the BBB prevents most small-molecule drugs and macromolecules from entering the brain across the listed CNS diseases. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [6] Patel M. M., Patel B. M., Crossing the blood–brain barrier: Recent advances in drug delivery to the brain. CNS Drugs 31, 109– 133 (2017). 28101766 sourceNot checked — no full text of the source

Disruption of the blood-brain barrier using osmotic, biochemical, or ultrasound techniques is effective to introduce drugs into the brain.

“Disruption of the BBB, using techniques such as osmotic disruption, biochemical disruption, and ultrasound-mediated disruption, is effective to introduce drugs into the brain; however, these transient BBB openings also allows for the leakage of plasma proteins into the brain, leading to neurotoxicity, vascular pathology, and chronic neuropathologic changes in the brain (1, 7, 11).”

• cites [1] Pardridge W. M., Blood–brain barrier delivery. Drug Discov. Today 12, 54– 61 (2007). 17198973 sourceNot checked — no full text of the source
• cites [7] Dong X., Current strategies for brain drug delivery. Theranostics 8, 1481– 1493 (2018). 29556336 PMC5858162 sourcePartly supported

  The Blood-brain tumor barrier section identifies hyperosmotic mannitol and bradykinin as ways to open the BBB, and the Non-invasive techniques section reports that ultrasound-assisted opening improved drug delivery in experimental studies. But the Brain permeability enhancers section says bradykinin-related results did not translate into a benefit in glioma patients, so the unqualified claim that all three techniques are effective overstates the evidence. (LLM, conf=0.85)

  Experimental — accuracy not yet measured
• cites [11] Villringer K., Cuesta B. E. S., Ostwaldt A.-C., Grittner U., Brunecker P., Khalil A. A., Schindler K., Eisenblätter O., Audebert H., Fiebach J. B., DCE-MRI blood–brain barrier assessment in acute ischemic stroke. Neurology 88, 433– 440 (2017). 28031392 sourceNot checked — no full text of the source

The blood-brain barrier prevents the transfer of most small-molecule drugs and macromolecules into the brain.

“it prevents the transfer of most of small-molecule drugs and macromolecules (e.g., peptides, gene drugs, and protein drugs) into the brain (1–6).”

• cites [1] Pardridge W. M., Blood–brain barrier delivery. Drug Discov. Today 12, 54– 61 (2007). 17198973 sourceNot checked — no full text of the source
• cites [2] Barchet T. M., Amiji M. M., Challenges and opportunities in CNS delivery of therapeutics for neurodegenerative diseases. Expert Opin. Drug Deliv. 6, 211– 225 (2009). 19290842 sourceNot checked — no full text of the source
• cites [3] Collins P. Y., Patel V., Joestl S. S., March D., Insel T. R., Daar A. S., Bordin I. A., Costello E. J., Durkin M., Fairburn C., Glass R. I., Hall W., Huang Y., Hyman S. E., Jamison K., Kaaya S., Kapur S., Kleinman A., Ogunniyi A., Otero-Ojeda A., Poo M.-M., Ravindranath V., Sahakian B. J., Saxena S., Singer P. A., Stein D. J., Anderson W., Dhansay M. A., Ewart W., Phillips A., Shurin S., Walport M., Grand challenges in global mental health. Nature 475, 27– 30 (2011). 21734685 PMC3173804 sourceCould not verify

  The List and Next Steps sections discuss research priorities and treatment of mental, neurological and substance-use disorders, but do not address whether the blood-brain barrier blocks drugs from entering the brain. Searches for the blood-brain barrier, small-molecule drugs, peptides and protein or gene drugs found no relevant passage in our copy. (LLM, conf=0.76)

  Experimental — accuracy not yet measured
• cites [4] Obermeier B., Daneman R., Ransohoff R. M., Development, maintenance and disruption of the blood-brain barrier. Nat. Med. 19, 1584– 1596 (2013). 24309662 PMC4080800 sourcePartly supported

  The Introduction says the BBB’s tight junctions and low transcytosis greatly limit movement of molecules into the brain. It does not specifically establish that most small-molecule drugs or macromolecular drugs, including the examples in the citing sentence, are prevented from entering. (LLM, conf=0.82)

  Experimental — accuracy not yet measured
• cites [5] Khorkova O., Wahlestedt C., Oligonucleotide therapies for disorders of the nervous system. Nat. Biotechnol. 35, 249– 263 (2017). 28244991 PMC6043900 sourcePartly supported

  The Abstract (page unavailable) says oligonucleotides do not readily cross the blood-brain barrier and that fewer than 1% of systemically delivered oligonucleotides reach the brain. “Assisted delivery and CNS targeting of ODNs” (page unavailable) also mentions small molecules with poor BBB permeability and intranasal delivery of peptides or proteins. The paper does not establish the broader claim that the BBB prevents transfer of most small-molecule drugs and macromolecules. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [6] Patel M. M., Patel B. M., Crossing the blood–brain barrier: Recent advances in drug delivery to the brain. CNS Drugs 31, 109– 133 (2017). 28101766 sourceNot checked — no full text of the source

Dimethyltryptamine (NT) has been reported to cross the BBB by active transport across the endothelial plasma membrane.

“The chemical structure of NT1 is based on the NT dimethyltryptamine, which has been reported to cross the BBB by active transport across the endothelial plasma membrane (21).”

• cites [21] Carbonaro T. M., Gatch M. B., Neuropharmacology of N,N-dimethyltryptamine. Brain Res. Bull. 126, 74– 88 (2016). 27126737 PMC5048497 sourceSupported

Dimethyltryptamine and other tryptamine derivatives cross the blood-brain barrier by active transport across the endothelial plasma membrane.

“For example, dimethyltryptamine and other tryptamine derivatives have been shown to cross the BBB by active transport across the endothelial plasma membrane (21).”

• cites [21] Carbonaro T. M., Gatch M. B., Neuropharmacology of N,N-dimethyltryptamine. Brain Res. Bull. 126, 74– 88 (2016). 27126737 PMC5048497 sourcePartly supported

  Section 2.2, “Accumulation and storage” (page not provided), states that DMT crosses the blood–brain barrier by active transport across the endothelial plasma membrane. It mentions other tryptamines as substrates for SERT and VMAT2, but does not say they cross the blood–brain barrier by that mechanism. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

ASO-mediated tau reduction has shown promising results in the treatment of Alzheimer's disease after local injection of Tau-ASO using an intracerebroventricular pump.

“because ASO-mediated tau reduction has shown promising results in the treatment of Alzheimer’s disease after the local injection of the Tau-ASO using an intracerebroventricular (ICV) pump (31, 32).”

• cites [31] DeVos S. L., Goncharoff D. K., Chen G., Kebodeaux C. S., Yamada K., Stewart F. R., Schuler D. R., Maloney S. E., Wozniak D. F., Rigo F., Bennett C. F., Cirrito J. R., Holtzman D. M., Miller T. M., Antisense reduction of tau in adult mice protects against seizures. J. Neurosci. 33, 12887– 12897 (2013). 23904623 PMC3728694 sourcePartly supported

  The Results section reports that Tau-ASO delivered by intracerebroventricular pumps reduced tau in adult mice. The Discussion describes treatment of Alzheimer’s disease as a possible future application, not a demonstrated result: this paper studied seizure protection, not treatment efficacy in Alzheimer’s disease. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites [32] DeVos S. L., Miller R. L., Schoch K. M., Holmes B. B., Kebodeaux C. S., Wegener A. J., Chen G., Shen T., Tran H., Nichols B., Zanardi T. A., Kordasiewicz H. B., Swayze E. E., Bennett C. F., Diamond M. I., Miller T. M., Tau reduction prevents neuronal loss and reverses pathological tau deposition and seeding in mice with tauopathy. Sci. Transl. Med. 9, eaag0481 (2017). 28123067 PMC5792300 sourcePartly supported

  The Results (‘Human Tau Antisense Oligonucleotides Reduce Human Tau mRNA in mice’ and ‘Tau ASO Treatment Prevents AT8-positive Tau Deposition’; page unavailable) report tau reduction and reduced pathology in PS19 mice. The Methods (‘Surgical Placement of Intracerebroventricular (ICV) Pumps and Tissue Collection’; page unavailable) confirm delivery by ICV pump. The Discussion calls this preclinical work a basis for a possible Alzheimer’s disease therapy; it does not report treatment results in people with Alzheimer’s disease. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

ASOs do not readily cross the blood-brain barrier, which remains a substantial obstacle for their clinical application in treating CNS disorders.

“However, the fact that ASOs do not readily cross the BBB remains one of the substantial obstacles for their clinical application in treating CNS disorders (28, 29).”

• cites [28] Rinaldi C., Wood M. J., Antisense oligonucleotides: The next frontier for treatment of neurological disorders. Nat. Rev. Neurol. 14, 9– 21 (2018). 29192260 sourceNot checked — no full text of the source
• cites [29] Talbot K., Wood M. J., Wrangling RNA: Antisense oligonucleotides for neurological disorders. Sci. Transl. Med. 11, eaay2069 (2019). 31554737 sourceSupported

Complicated modifications are always needed to ensure that nanoparticles produced are blood-brain barrier-permeable.

“Crossing the BBB with various nanoparticles, such as liposomes, cationic polymers, inorganic nanoparticles, and nanocapsules, has shown promise in delivery of various cargos into the CNS, but complicated modifications are always needed to ensure that the particles produced are BBB-permeable (15, 18, 19).”

• cites [15] Zhou Y., Peng Z., Seven E. S., Leblanc R. M., Crossing the blood-brain barrier with nanoparticles. J. Control. Release 270, 290– 303 (2018). 29269142 sourceNot checked — no full text of the source
• cites [18] Blanco E., Shen H., Ferrari M., Principles of nanoparticle design for overcoming biological barriers to drug delivery. Nat. Biotechnol. 33, 941– 951 (2015). 26348965 PMC4978509 sourcePartly supported

  The Opsonization/sequestration section reports that polysorbate 80–coated nanoparticles enhanced drug delivery across the blood-brain barrier. That supports an example of a modification that aided crossing, but the paper does not say complicated modifications are always necessary to make nanoparticles BBB-permeable. (LLM, conf=0.85)

  Experimental — accuracy not yet measured
• cites [19] Wen J., Wu D., Qin M., Liu C., Wang L., Xu D., Vinters H. V., Liu Y., Kranz E., Guan X., Sun G., Sun X., Lee Y. J., Martinez-Maza O., Widney D., Lu Y., Chen I. S. Y., Kamata M., Sustained delivery and molecular targeting of a therapeutic monoclonal antibody to metastases in the central nervous system of mice. Nat. Biomed. Eng. 3, 706– 716 (2019). 31384008 PMC6736720 sourcePartly supported

  The Abstract (page unavailable) describes difficulties with existing CNS delivery vehicles and says substantial improvements are needed, while Results and discussion, “Nanocapsules facilitate CNS penetration” (page unavailable), describes an engineered nanocapsule designed for brain delivery. Neither passage establishes that complicated modifications are always necessary for every nanoparticle to cross the BBB; that universal requirement overstates the paper. (LLM, conf=0.85)

  Experimental — accuracy not yet measured

AmB cannot be used clinically for the treatment of brain fungal infections due to its BBB impermeability.

“However, it cannot be used clinically for the treatment of brain fungal infections due to its BBB impermeability (25, 26).”

• cites [25] Ostrosky-Zeichner L., Marr K. A., Rex J. H., Cohen S. H., Amphotericin B: Time for a new “gold standard”. Clin. Infect. Dis. 37, 415– 425 (2003). 12884167 sourceNot checked — no full text of the source
• cites [26] Xu N., Gu J., Zhu Y., Wen H., Ren Q., Chen J., Efficacy of intravenous amphotericin B-polybutylcyanoacrylate nanoparticles against cryptococcal meningitis in mice. Int. J. Nanomedicine 6, 905– 913 (2011). 21720503 PMC3124396 sourcePartly supported

  The Introduction says BBB impermeability limits the clinical application of AmB against cryptococcal meningitis, supporting the stated reason. But the claim's absolute wording—“cannot be used clinically”—goes beyond the paper: the Discussion describes intrathecal injection as a clinical approach developed to overcome that limitation. (LLM, conf=0.87)

  Experimental — accuracy not yet measured

Previous experience with biomacromolecules indicated that a slightly longer tail length would be more appropriate for the NT1-O14B choice.

“our previous experience with biomacromolecules (22) indicated that the slightly longer tail length would be more appropriate here.”

• cites [22] Wang M., Zuris J. A., Meng F., Rees H., Sun S., Deng P., Han Y., Gao X., Pouli D., Wu Q., Georgakoudi I., Liu D. R., Xu Q., Efficient delivery of genome-editing proteins using bioreducible lipid nanoparticles. Proc. Natl. Acad. Sci. U.S.A. 113, 2868– 2873 (2016). 26929348 PMC4801296 sourceSupported

In a previous study, ~80% reduction of tau mRNA and ~70% reduction of tau protein were achieved by continuously dosing 28 days with 25-μg Tau-ASO per day through local injection by ICV pump.

“Notably, in the previous study, ~80% reduction of tau mRNA and ~70% reduction of tau protein were achieved by continuously dosing 28 days, and the dose was 25-μg Tau-ASO per day through local injection by ICV pump (31).”

• cites [31] DeVos S. L., Goncharoff D. K., Chen G., Kebodeaux C. S., Yamada K., Stewart F. R., Schuler D. R., Maloney S. E., Wozniak D. F., Rigo F., Bennett C. F., Cirrito J. R., Holtzman D. M., Miller T. M., Antisense reduction of tau in adult mice protects against seizures. J. Neurosci. 33, 12887– 12897 (2013). 23904623 PMC3728694 sourcePartly supported

  The Methods describe 28-day ICV osmotic pumps, and the Results section on endogenous tau expression reports that 25 μg/day Tau ASO-3 reduced tau mRNA by more than 75%, with a reduction exceeding 80% still present four weeks after infusion stopped. The Results also report reduced tau protein after this treatment, but our copy does not give the claimed approximately 70% protein reduction. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

This AmB concentration in the brain is higher than that which has previously been reported.

“Notably, this concentration of AmB is higher than that which has previously been reported (26).”

• cites [26] Xu N., Gu J., Zhu Y., Wen H., Ren Q., Chen J., Efficacy of intravenous amphotericin B-polybutylcyanoacrylate nanoparticles against cryptococcal meningitis in mice. Int. J. Nanomedicine 6, 905– 913 (2011). 21720503 PMC3124396 sourceSupported

Tryptamine-derived lipidoid-doped lipid nanoparticle formulations facilitate cargos crossing the blood-brain barrier.

“Tryptamine-derived lipidoid-doped lipid nanoparticle formulations facilitate cargos crossing the blood-brain barrier.”

The previously published synthetic lipids 76-O16B, EC16-80, and 113-O16B were not effective in delivering DiR into the mouse brain by themselves.

“We found that none of these lipids were effective in delivering the DiR into the mouse brain by themselves; however, after doping these lipids with the NT1-O12B, strong DiR signals could be observed in the mouse brain (fig. S4).”

Mice treated with Tau-ASO formulated in NT1-O14B/306-O12B-3 (3:7, w/w) showed substantially reduced total tau protein level compared with the untreated group.

“Comparing with the untreated group, mice treated with Tau-ASO formulated in NT1-O14B/306-O12B-3 (3:7, w/w) showed substantially reduced total tau protein level.”

Delivery of GFP-Cre resulted in induction of red fluorescence in neurons using a floxed tdTomato mouse line (Ai14).

“Delivery of both Tau-ASOs and GFP-Cre resulted in functional intracellular delivery, as indicated by a reduction in both tau mRNA and protein levels in the case of ASO delivery, and the induction of red fluorescence in neurons using a floxed tdTomato mouse line (Ai14) in the case of GFP-Cre delivery.”

NT1-O12B showed the highest DiR fluorescence intensity among all NT-lipidoids.

“We chose NT1-O12B as the dopant for enhanced brain delivery because it showed the highest DiR fluorescence intensity (Fig. 1C) among all NT-lipidoids.”

In GFP-HEK cells, GFP silencing was observed when the 306-O12B-3 doping ratio was greater than 50% (5:5 weight ratio or more in favor of 306-O12B-3).

“When the doping ratio of 306-O12B-3 was greater than 50% (i.e., 5:5 weight ratio or more in favor of 306-O12B-3), we observed the GFP silencing in GFP-HEK cells”

AmB formulated in NT-lipidoid formulation showed an opaque solution.

“However, the AmB formulated in NT-lipidoid formulation showed opaque solution (fig. S7A), indicating the large size of the particles in the solution.”

Changes in the chemical structure of the NT1-lipidoid affected the DiR signal detected in the mouse brain.

“We found that the change in the chemical structure of the NT1-lipidoid affected the DiR signal detected in the brain, indicating that the successful delivery of DiR into the brain is highly dependent on the specific chemical structure.”

The authors demonstrated a simple and effective approach for delivering cargos into the brain using neurotransmitter-derived synthetic lipids.

“Here, we demonstrated a simple and effective approach for delivering cargos into the brain using neurotransmitter (NT)–derived synthetic lipids.”

Dynamic light scattering and transmission electron microscopy indicated that NT-lipidoids self-assembled into spherical liposome-like structures.

“these structures indeed self-assembled into spherical liposome-like structures (fig. S2).”

NT2- and NT3-derived lipidoids with any linkers produced no strong DiR signals in the mouse brain.

“No strong DiR signals were observed from the mouse brain treated with NT2- and NT3-derived lipidoids with any linkers.”

The NT-lipidoid AmB nanoparticles had hydrodynamic diameters in the range of 750 to 800 nm.

“DLS results (fig. S7B) showed that the nanoparticles are in range of 750 to 800 nm in diameter.”

Scrambled ASO delivered by Lipofectamine 2000 showed no GFP silencing.

“Scrambled ASO delivered by Lipofetamine 2000 showed no GFP silencing”

NT1-O14B LNPs alone showed no GFP silencing effect in HEK-GFP cells.

“The NT1-O14B alone showed no GFP silencing effect (10:0 ratio in Fig. 3B)”

GFP silencing efficiency increased as the 306-O12B-3 doping ratio increased.

“silencing efficiency increasing as the 306-O12B-3 doping ratio increased.”

Doping NT1-lipidoid into 306-O12B-3 LNPs led to successful gene silencing in vitro.

“while doping NT1-lipidoid into 306-O12B-3 LNPs led to successful gene silencing in vitro.”

Among the tested NT1-O12B/PBA-Q76-O16B ratios, the 3:7 (w/w) ratio DiR-loaded LNPs provided the strongest fluorescent signal in the mouse brain.

“Using DiR as a cargo, we observed that lipidoids containing NT1-O12B and PBA-Q76-O16B at a 3:7 (w/w) ratio provided the strongest fluorescent signal in mouse brain when compared with all other lipid ratios (Fig. 2D).”

• AssumptionRests on an assumption: DiR fluorescence intensity in the mouse brain is proportional to the amount of DiR delivered across the BBB.

No tau mRNA silencing was observed in mixed lipidoid formulations at other ratios (7:3 and 1:9).

“No tau mRNA silencing was observed in the mixed lipidoid formulations in other ratios (i.e., 7:3 and 1:9).”

NT1-lipidoid can be doped into otherwise blood-brain barrier-impermeable lipid formulations.

“The NT1-lipidoid can be doped into otherwise BBB-impermeable lipid formulations, and the resulting lipid nanoparticles (NT1-LNPs) also gained the ability to cross the BBB.”

Doping the previously published synthetic lipids 76-O16B, EC16-80, and 113-O16B with NT1-O12B resulted in strong DiR signals in the mouse brain.

“We found that none of these lipids were effective in delivering the DiR into the mouse brain by themselves; however, after doping these lipids with the NT1-O12B, strong DiR signals could be observed in the mouse brain (fig. S4).”

The authors developed a new, simple, and effective brain delivery system.

“In summary, we developed a new, simple, and effective brain delivery system, which may find wide applications in treating CNS diseases or provide tools to study the brain function.”

When ASO was delivered using either pure NT1-O14B or pure 306-O12B-3, no tau mRNA reduction in the brain tissues was detected.

“when ASO was delivered using either pure NT1-O14B or pure 306-O12B-3, no tau mRNA reduction in the brain tissues was detected.”

The DiR fluorescence signal intensity in the mouse brain at the 3:7 ratio was 4.5-fold higher than that of brain treated with DiR formulated in pure NT1-O12B.

“The fluorescent signal intensity at a 3:7 ratio was 4.5-fold higher than that of brain treated with DiR formulated in pure NT1-O12B (fig. S8).”

• AssumptionRests on an assumption: DiR fluorescence intensity in the mouse brain is proportional to the amount of DiR delivered across the BBB.

Tryptamine-derived lipidoids (NT1-lipidoids) could carry fluorescent dye to cross the blood-brain barrier efficiently without any additional targeting ligands.

“We found that the tryptamine-derived lipidoids (NT1-lipidoids) could carry fluorescent dye to cross the BBB efficiently without any additional targeting ligands.”

The resulting NT1-lipid nanoparticles gained the ability to cross the blood-brain barrier.

“The NT1-lipidoid can be doped into otherwise BBB-impermeable lipid formulations, and the resulting lipid nanoparticles (NT1-LNPs) also gained the ability to cross the BBB.”

Among NT1-lipidoids, shorter aliphatic tail chain length results in greater DiR fluorescence intensity in the mouse brain.

“We also observed that the length of the aliphatic tail chain notably influenced the observed fluorescent intensity, in which NT1-lipidoids containing a shorter aliphatic chain length result in a greater fluorescence intensity (fig. S3).”

In mice, 24 h after intravenous injection of AmB (5 mg/kg) in pure NT1-lipidoid formulations (NT1-O12B, NT1-O14B, NT1-O16B, and NT1-O18B), the brain AmB concentration was around 150 ng/g tissue.

“As shown in fig. S6, the AmB concentration in the brain tissue of all the four groups was around 150 ng/g (AmB/tissue).”

Delivery of Tau-ASOs resulted in a reduction in both tau mRNA and protein levels.

“Delivery of both Tau-ASOs and GFP-Cre resulted in functional intracellular delivery, as indicated by a reduction in both tau mRNA and protein levels in the case of ASO delivery, and the induction of red fluorescence in neurons using a floxed tdTomato mouse line (Ai14) in the case of GFP-Cre delivery.”

Without doping with NT1-lipidoid, AmB was nearly undetectable in the brain after intravenous injection of pure PBA-Q76-O16B/AmB.

“Without doping with NT1-lipidoid, the AmB was nearly undetectable in the brain after intravenous injection of pure PBA-Q76-O16B/AmB.”

Doping NT1-lipidoids to otherwise BBB-impermeable lipidoids resulted in the functional delivery of both ASO and Cre protein into the brain.

“Doping NT1-lipidoids to otherwise BBB-impermeable lipidoids also resulted in the functional delivery of both ASO and Cre protein into the brain.”

Doping NT1-lipidoids into BBB-impermeable LNPs resulted in LNPs that can cross the BBB effectively.

“Doping the NT1-lipidoids into BBB-impermeable LNPs resulted in LNPs that can cross BBB effectively.”

The concentration of AmB in the brain remains low compared with the concentration of AmB in other organs, such as liver and spleen.

“the concentration in brain remains low compared with the concentration of AmB in other organs, such as liver and spleen (fig. S9D).”

The delivery of ASO and Cre protein into the brain was functional.

“It is notable that the delivery of these biomacromolecules was functional.”

The red fluorescence signal from cells in the hippocampus was weaker than that in the cerebral cortex and cerebellum.

“The red fluorescence signal from cells in hippocampus is weaker than that in cerebral cortex and cerebellum”

In this study, ~50% reduction of tau mRNA and ~30% reduction of tau protein were achieved by only five times intravenous injection of 20-μg Tau-ASO/LNP complex.

“In comparison, ~50% reduction of tau mRNA and ~30% reduction of tau protein were achieved by only five times intravenous injection of 20-μg Tau-ASO/LNP complex in this study.”

No tdTomato expression in the brain was observed for mice injected with LNP formulations using either pure NT1-O14B (10:0) or pure PBA-Q76-O16B (0:10).

“In contrast, no tdTomato expression in the brain was observed for the mice injected with LNP formulations using either pure NT1-O14B (10:0) or pure PBA-Q76-O16B (0:10).”

Doping NT1-O12B to the BBB-impermeable lipidoid PBA-Q76-O16B resulted in an AmB formulation that could cross the BBB.

“Doping NT1-O12B to the BBB-impermeable lipidoid PBA-Q76-O16B resulted in an AmB formulation that could cross BBB.”

The NT1-lipidoid can serve as a new BBB-penetrating moiety.

“We found that the NT1-lipidoid can serve as a new BBB-penetrating moiety.”

Strong tdTomato signals were observed in multiple regions of the brain, including cerebral cortex, hippocampus, and cerebellum, in Ai14 mice treated with (−27)GFP-Cre complexed with NT1-O14B:PBA-Q76-O16B = 3:7 LNPs.

“As shown in Fig. 4B, strong tdTomato signals were observed in multiple regions of the brain, including cerebral cortex, hippocampus, and cerebellum.”

• Also stated in: § Delivery of GFP-Cre fusion protein for gene recombination in the Ai14 mouse brain · hedged

  “After 3 weeks, the group of NT1-O14B:PBA-Q76-O16B = 3:7 showed tdTomato expression indicative of Cre-mediated recombination in cerebral cortex, hippocampus, and cerebellum.”

• Also stated in: § DISCUSSION · plain

  “the genome editing was observed in various different regions of the mouse brain, including cerebral cortex, hippocampus, and cerebellum.”

The physical properties (size, zeta potential, and PDI) of NT1 and NT2 lipidoids were very similar.

“Notably, the physical properties of the NT1 and NT2 lipidoids such as size, zeta potential, and PDI were very similar (fig. S2)”

At intermediate weight ratios, successful tau knockdown indicates that ASO can be delivered across both the blood-brain barrier and the cell membrane.

“At the intermediate weight ratios, we see successful tau knockdown, indicating that the ASO can be delivered across both the BBB and the cell membrane.”

The specific structure of the NT-lipidoid drives its ability to cross the BBB.

“These results support our hypothesis that the specific structure of the NT-lipidoid is driving the ability to cross the BBB, although the specific mechanism for NT-lipidoid–mediated brain delivery is not known yet.”

• AssumptionRests on an assumption: DiR fluorescence signal in the mouse brain after intravenous injection reflects delivery of DiR across the BBB into brain tissue rather than residual intravascular DiR.
• AssumptionRests on an assumption: The physical properties of NT2- and NT3-lipidoids are similar enough that the difference in DiR brain signal between NT1 and NT2/NT3 lipidoids is due to BBB crossing rather than differences in particle properties.

In a previous study, none of the authors' synthetic lipidoid nanoparticles were capable of permeating the BBB to deliver AmB into the mouse brain.

“Recently, we formulated AmB in synthetic lipidoid nanoparticles and conducted a thorough pharmacokinetics (PK) and biodistribution study of the AmB formulations using traditional synthetic LNPs, but in that study, none of our LNPs were capable of permeating the BBB to deliver AmB into the mouse brain (27).”

• cites [27] Liu F., Yang L., Li Y., Junier A., Ma F., Chen J., Han H., Glass Z., Zhao X., Kumamoto C. A., Sang H., Xu Q., In vitro and in vivo study of Amphotericin B formulation with quaternized bioreducible lipidoids. ACS Biomater Sci. Eng. 6, 1064– 1073 (2020). 33464849 sourceNot checked — no full text of the source

In a previous report, AmB was undetectable in the brain after systemic delivery with traditional synthetic lipidoids.

“Notably, in our previous report, AmB was undetectable in the brain after systemic delivery with traditional synthetic lipidoids, indicating that the NT1-lipidoid formulations enhanced the AmB delivery into the mouse brain.”

• cites [27] Liu F., Yang L., Li Y., Junier A., Ma F., Chen J., Han H., Glass Z., Zhao X., Kumamoto C. A., Sang H., Xu Q., In vitro and in vivo study of Amphotericin B formulation with quaternized bioreducible lipidoids. ACS Biomater Sci. Eng. 6, 1064– 1073 (2020). 33464849 sourceNot checked — no full text of the source

Finding the optimal doping ratio is important for brain delivery.

“These results showed the key role of NT1-lipidoids in facilitating the brain delivery and the importance of finding the optimal doping ratio.”

Tau knockdown is specifically due to sequence-specific ASO silencing.

“demonstrating that the tau knockdown is specifically due to sequence-specific ASO silencing.”

In a previous report, quaternized lipidoids provided stable AmB formulations with smaller particle size compared with nonquaternized lipids.

“In the previous report, we found that the quaternized lipidoids provided stable AmB formulation with smaller particle size, comparing with the nonquaternized lipids (27).”

• cites [27] Liu F., Yang L., Li Y., Junier A., Ma F., Chen J., Han H., Glass Z., Zhao X., Kumamoto C. A., Sang H., Xu Q., In vitro and in vivo study of Amphotericin B formulation with quaternized bioreducible lipidoids. ACS Biomater Sci. Eng. 6, 1064– 1073 (2020). 33464849 sourceNot checked — no full text of the source

Some neurotransmitters have been demonstrated to cross the blood-brain barrier.

“Notably, some NTs have been demonstrated to cross the BBB (20).”

• cites [20] Snowman A. M., Snyder S. H., Cetirizine: Actions on neurotransmitter receptors. J. Allergy Clin. Immunol. 86, 1025– 1028 (1990). 1979798 sourceNot checked — no full text of the source

Successful delivery of DiR into the brain is highly dependent on the specific chemical structure of the NT-lipidoid.

“We found that the change in the chemical structure of the NT1-lipidoid affected the DiR signal detected in the brain, indicating that the successful delivery of DiR into the brain is highly dependent on the specific chemical structure.”

• AssumptionRests on an assumption: DiR fluorescence intensity in the mouse brain is proportional to the amount of DiR delivered across the BBB.

The results for AmB delivery closely matched the results for DiR delivery across the tested formulations.

“Thus, the results for AmB delivery closely matched the results for DiR delivery (Fig. 2, D and E).”

• AssumptionRests on an assumption: DiR fluorescence intensity in the mouse brain is proportional to the amount of DiR delivered across the BBB.

NT1-lipidoid formulations enhanced AmB delivery into the mouse brain compared with traditional synthetic lipidoids.

“Notably, in our previous report, AmB was undetectable in the brain after systemic delivery with traditional synthetic lipidoids, indicating that the NT1-lipidoid formulations enhanced the AmB delivery into the mouse brain.”

This is the first observation of effective genome recombination in neuronal cells of the Ai14 tdTomato mouse brain through intravenous injection of Cre protein delivery vehicles.

“This is the first observation of effective genome recombination in neuronal cells of the Ai14 tdTomato mouse brain through intravenous injection of Cre protein delivery vehicles.”

The resulting NT-lipidoids are amphiphilic and thus should be capable of self-assembly into either micelles or liposomes when prepared in aqueous solution.

“The resulting NT-lipidoids are amphiphilic and thus should be capable of self-assembly into either micelles or liposomes when prepared in aqueous solution.”

The GFP silencing was ASO sequence-specific.

“indicating that the GFP silencing was truly ASO sequence–specific.”

NT1-lipidoids play a key role in facilitating brain delivery of AmB.

“These results showed the key role of NT1-lipidoids in facilitating the brain delivery and the importance of finding the optimal doping ratio.”

• AssumptionRests on an assumption: DiR fluorescence signal in the mouse brain after intravenous injection reflects delivery of DiR across the BBB into brain tissue rather than residual intravascular DiR.
• AssumptionRests on an assumption: DiR fluorescence intensity in the mouse brain is proportional to the amount of DiR delivered across the BBB.

NT1-O14B alone is not effective for delivering ASO intracellularly.

“indicating that this lipidoid alone is not effective for delivering ASO intracellularly.”

This is the first report of effective genome editing in neuronal cells of the Ai14 mouse brain through intravenous injection of Cre protein delivery vehicles.

“Furthermore, this is the first report of effective genome editing in neuronal cells of the Ai14 mouse brain through intravenous injection of Cre protein delivery vehicles”

The NT1-lipidoid-doped LNP system is a new, simple, universal, and effective brain delivery system.

“In summary, we developed a new, simple, universal, and effective brain delivery system”

The difference in delivery capability between NT1 and NT2 lipidoids is likely a result of chemical rather than physical properties.

“indicating that difference in delivery capability is likely a result of chemical rather than physical properties.”

Engineering LNPs to balance the need to cross both the BBB and the cell membrane is important to achieve successful gene recombination in neuronal cells upon systemic administration.

“These results again demonstrated the importance of engineering the LNPs to balance the need to cross both the BBB and the cell membrane to achieve the successful gene recombination in neuronal cells upon systemic administration.”

• AssumptionRests on an assumption: Functional delivery of ASO or Cre protein into the brain requires crossing two barriers: the blood-brain barrier and the cell membrane barrier.

Even if functional heads themselves can cross the BBB, their derived lipidoids may not.

“The results indicated that even if the functional heads themselves can cross BBB, their derived lipidoids may not (Fig. 1C).”

Systemic delivery has the advantage over local injection to reach a wide area in the brain.

“The successful gene recombination was observed in multiple regions in the mouse brain, which showed the advantage of systemic delivery over local injection to reach a wide area in the brain.”

• Also stated in: § Delivery of GFP-Cre fusion protein for gene recombination in the Ai14 mouse brain · plain

  “The successful gene recombination was observed in multiple regions in the mouse brain, which showed the advantage of systemic delivery”

Doping with the same NT1-lipidoid for each delivery application demonstrates a unique simplicity and flexibility of this system.

“It is particularly notable that, while each delivery application used a different BBB-impermeable LNP carrier, we simply doped with the same NT1-lipidoid for each application. This demonstrates a unique simplicity and flexibility of this system.”