TECHNOLOGY EXPLAINER
Lipid Nanoparticles (LNPs)
— Performance depends on lipid pKa and how the particles are mixed
LNP is a particle made of four types of lipids. The central ionized lipids,The effect changes with a digit just by 0.5 drop of pKais shown in a paper comparing 56 types of lipids. and the process of making particles"Mix two liquids"Closedesign and mixing conditions are standard items of the product as it isMaterial
LNP as a formulation of mRNA vaccine, design of ionized fats, ratio of fats, production process, and delivery to other than the liver(The role of each component, the composition of one time, and the cold chain) is this seriesmRNAContact Us This article is so serious about it,design of ionized fats, ratio of fats, production process, and delivery to other than the liverFilter by The chemical of the nucleic acid itself is "nucleic acid medicine" and the contract manufacturing of the original medicine is "CDMO".
- What is LNP?
- Ionizing lipids - pKa determines the effect
- Material Technician Perspective 1: pKa is not “contained”
- Molar ratio of 4 components - 40/10/40/10 to 50/10/38.5/1.5
- ulation of this paper: reverse the ratio of fats from the composition of approved products
- PEG lipids are part of "premise to be removed"
- How to make: From T-shaped mixing to micro flow path
- Manufacturing process and standard of approved products - read from EPAR
- Perspective of Material Technicians 2: Standard items are required specifications of materials as they are
- Pathogens in the body: ApoE escapes only 1 to 2% with endsomes
- Delivering to other than the liver: Recent steps
- Issues and Undetermined
- Glossary/Reference Materials/Reference Table
Facts= Contents described in the publication material (with source link)
The calculation of this paper= The value calculated based on the specified premise
Undetermined / Future= Items that have not been confirmed by the research stage and plan
In addition to this, it is possible to organize structures and read materials and process designs."Description"is distinguished.
About this articleExplanation of material technology and manufacturing technologyComment It does not evaluate the effects and safety of treatments or vac s, and is not medical advice. The approval or the composition of the product is only quoted in the published material of the regulatory authority (FDA/ ). The number of animal experiments does not apply to humans as it is.
1. What is LNP?
- What to do:Nucleic acid (siRNA or mRNA) is decom by the body and is not contained in cells. LNP ItCarrying into lipid particlesMaterials for Copyright © Onpattro. All Rights Reserved.“Meeting with siRNA to protect against immediate decomposition in the circulatory system and to help deliver to the target part of the liver”Contact UsFacts
- What's done:Ionized lipids, lipid lipids (DSPC), cholesterol, PEG lipids4 ingredientsFactsHome For approved products,Ionized fat and PEG fat are "New additives"Reviewed asFacts
- Is it different from liposome?TEM report from cryo-TEM image"The particles of patisiran formulations are spherical nanoparticles with high electronic density cores, which do not have a typical double-membrane structure in regular liposomes.Facts。"Granule packed with contents" instead of "hollow bag"Home
The performance of LNP is determined by the pKa of ionized lipids first from the compound ratio of lipids. Jayaraman compares 56 types of amino lipids,Strongly correlate the strength of pKa and gene suppression, the optimal range is pKa 6.2 to 6.5 I'm reporting thatFactsHome This isProblems with design rather than compound designandValue to measure after particle(This article explains).
2. Ionizing lipids – pKa determines the effect
ionized fatNo charge in the vicinity of neutrality.Lipids This report explains the meaning of this natureFacts。
"Ionizing aminolipid DLin-MC3-DMA gives clear physical and chemical properties that control the formation of particles, siRNA interaction, cell uptake, fusogenicity (the ability to combine with endosomal membranes), and the release from siRNA endosomes. This lipid was chosen after extensively reviewing the relevant ionic lipids.Facts
Facts
DLin-MC3-DMA entityFormula C43H79NO2, Weight 642.09、Two linole chains (C18:2) with double bonds, colorless to light yellow liquidHomeWell melted into organic solvents and hardly melted into waterFacts。
How good is pKa? Jayaraman et al. 2012 56 types of aminolipidsSynthesizes and suppresses mouse VII factor (FVII) genes (ED)50) and pKaFacts。
| Paper Description | |
|---|---|
| Close | “A strong correlation between lipid pKa and mouse FVII gene suppression (FVII ED50) was found, and the optimal pKa range was 6.2–6.5.” |
| How to measure pKa | TNS(2-(p-toluidino)-6-naphthalene sulfonic acid)0.5 engraving from pH 3 to 10Twisted and measured. TNSThe meeting occurs when a lipid nanoparticle (LNP) encounters a positively charged membrane surface, and it does not occur when it is free in the solution. |
| Structure and pKa | Distance between ester and amine1 to 5 pieceschange pKa4.17〜7.16Changed in range |
| Most active lipids | DLin-MC3-DMA(lipid 16): pKa 6.44, ED50 0.03 mg/kgHome When pKa is shifted to acidic side and base side, the activity decreased greatly |
| Condition for comparison | ParticleMax 3 times ex ded with 80 nm membraneParticle size70〜90 nmMulti-dispersion0.11±0.04（n＝56）Contact Us siRNA/Total fat weight ratio is about0.05 |
AllFacts(Jayaraman et al 2012 [Reference 1]).Mouse resultsand not human.
3. Perspective of material ians 1: pKa is not “contained”
If it is a formulation material, it does not reach the object of the targetof the mixing ratioYou can use it. ionized fat pKa,The properties of the changes on N s, stretching one chain between esters and aminesHome Jayaraman Home As a result, pKa moves from 4.17 to 7.16DutchRe skeletonCommentFactsFacts。
In other words"I want to raise pKa only 0.3" will be changed as it isThat is This article explains. Compared with 56 types,Structural-Physical correlation to be filled with experimentsTypical material development.
Another important thing isContact UsHOME TNS methodAfter making a particle, examine the positive charge of the membrane by pH titrationHow toFacts。 The pKa of the particle surface is measured instead of the dissociation constant.Dutch This isMeasuring the zeta potential of fluoropolymers and acidity of resinsIt is the same idea as This article explains. not material packaging,When usedto manage.
Therefore, we thoroughly understand the condition of the comparison experiment.Up to 3 times ex ded with 80 nm membrane, particle diameter 70-90 nm, multi dispersion 0.11±0.04only pKaFacts。 The effect of pKa is invisible while the particle size is brokenIt is also (explanation by this article).
4. Molar ratio of 4 components: 40/10/40/10 to 50/10/38.5/1.5
When pKa is determined,How to mix 4 ingredientsComment This transition is clearly written in the papers of Jayaraman.
The standard composition of the comparative experiment is amino lipids/DSPC/Cholesterol/PEG lipidsMolar Ratio 40/10/40/10 CommentFacts。
、“The ratio of four components (50/10/38.5/1.5) has been further optimized to increase the activity of in vivo.”HOME Mouse FVII modelED50 0.005 mg/kg Achieve this6x improvement compared to 40/10/40/10 compositionFactsHome Tests with the same lipid composition in canicusFacts。
Note:How to changeComment Ionized fat is 40 to 50 %, PEG lipids are reduced from 10 1.5% to 1.5 1.5%, less than one- rdHome This is handled in Chapter 6.
5. Calculation of this paper: reverse the ratio of the molar from the composition of the approved product.
The ratio of molars optimized by the paper isIs it used in actual approved products?——It is confirmed from the description of the attachment.
- patisiran（siRNA）：2 mg(equivalent to 2.1 mg as patisiran sodium salt)
- DLin-MC3-DMA：13.0 mg
- Cholesterol:6.2 mg
- DSPC：3.3 mg
- PEG2000-C-DMG：1.6 mg
- Potassium Phosphate 0.2 mg/ Sodium Chloride 8.8 mg/ Hydrogen Phosphate 2.3 mg/ Injectable water.pH about 7.0
- patisiran sodium salt formula C412H480N148Na40O290P40, weight 14,304 Da
FDA AttachmentsFactsHome DLin-MC3-DMA 642.09PEG2000-C-DMG 2,650±300 Da() Public Examination Report)FactsHome Cholesterol 386.7DSPC 790.1（PubChem）FactsComment
Re the mass to the number of mors and seek the ratio of morsThe calculation of this paper。
- DLin-MC3-DMA：13.0 ÷ 642.09 ＝ 20.2 µmol
- Cholesterol: 6.2 ÷ 386.7 = 16.0 µmol
- DSPC：3.3 ÷ 790.1 ＝ 4.18 µmol
- PEG2000-C-DMG：1.6 ÷ 2,650 ＝ 0.60 µmol
- Total 40.99 μ。. Molar action Ionized fat 49.3% / DSPC 10.2% / cholesterol 39.0% / PEG fat 1.5%
This corresponds to 50/10/38.5/1.5 which the paper is "optimized"The calculation of this paper。
Other conversionsThe calculation of this paper。
- Weight ratio of lipids/siRNA:Total fat 6.2+13.0+3.3+1.6 = 24.1 mg. 24.1 ÷ 2 = About 12 times
- Weight ratio of siRNA/Total lipids:2 ÷ 24.1 ＝ 0.083(Comparison of papers is about ())
- siRNA Ionizing Lipids per :siRNA is 2 mg ÷ 14,304 = 0.140 μ。. 20.2 ÷ 0.140 = 145 molecules
- Ratio of amine/phosphate:The negative charge of siRNA one is about 40 because of the si type of P. 145 ÷ 40 = about 3.6
Premises and Limits:PEG2000-C-DMG2,650±300Because it is a wide value, its molar percentage also removes the width (2,350 and 1.7%, 2,950). Cholesterol weight of cholesterol and DSPC is from PubChem. The ratio of amine/phosphateIonized lipids contain one amine per and assumed that everything is electrically chargedWhen the upper limit is Actual static rate depends on pH and pKaHome It does not indicate the manufacturing yield ratio.
6. PEG lipids are part of "premise to be removed"
As seen in Chapter 4, PEG lipids10 % to 1.5 %Cancel Why did you need to reduce? The role is clearly written in our report.
PEG Fat Chain Length"The PEG lipids from the circulating particles are dissolved over time, and the particles without PEG appear, which is taken into the liver cells, leading to the release of siRNA"SelectedFacts。
PEG2000-C-DMGformula (C2H4O)n C36H73NO5 (n about 47), weight 2,650±300 Da, white to ash white powderHomesoluble in water, chloroform and acetonitrileFacts。
"Outing" is built in the designPEG is fat. The speed to be removedLength of hydrophobic chainHome Mui et al. conducted a quantitative examinationFacts。
Dialkyll ChainCarbon Number 14, 16, 18About three types of PEG lipidsRapid transfer from LNP to plasma lipoproteinin vivoFacts。
“1.5 1.5% PEG lipids are threshold concentrations that minimize the influence of chain length on gene suppression in the liver, but still the amount and distribution of LNP drugs can be changed.”Facts
"When the concentration is increased to 2.5% and 3.5%, the gene suppression of liver cells is greatly impaired in the PEG lipids with a C18 chain, but the shorter dimyristyl (C14) chain has little effect."Facts
The same paper is PEG lipids"It is necessary to control the process of self-organization when lipid particles are formed.""Transportation to Liver Cells and Negative Effects on Genetic Control in Liver Cells"Contact UsFacts。
PEG lipid position is special."When creating a particle, it is necessary to stop self-organization and determine the particle size."Home HomeAfter entering the body, it prevents cells from being importedFacts。
The answer was taken there."Re」 the amount" and "Easy to out"Comment PEG2000-C-DMGDimythyl(C14) ChainHomeFactsHome MuiC14 chains are less likely to be suppressed even if they increase the concentration = fasterHomeFacts。
For engineers dealing with dispersants and emulsifiers,"It is necessary for film formation, but it is not necessary to remain in the final product"Not unusual. HomeVolatile / GrilledYou can use it.in the bodyHome HomeDesign detachable speed with the length of the hydrophobic chain——"The removal process is embedded in the structure side."(This article explains)
7. How to make: From T-shaped mixing to micro flow path
LNP ManufacturingMix "Ethanol phase melted fat" and "water phase melted nucleic acid"Process is central. This mixer has changed in 20 years.
| Generation | What is obtained (paper description) | |
|---|---|---|
| Jeffs （2005） | T-shaped mixing roomDNA buffer liquid and ethanol – Convey the flow of lipids and lower the ethanol concentration at once.No Ex sionHow to expand and produce | Particle sizeLess than 200 nma single decentralized follicle. DNAMore than 80%Home cryo-TEMMixing single layer, double layer and multi layer |
| Belliveau （2012） | Micro flow channel mixingHome Nanol scalemsAllow mixing | 20 nm or moreLNP of "Limit Size" in a wide range of conditionsNearly complete siRNA e tion raterate well. Multi-dispersibilityMinimum 0.02[Home] by mouse liver cellssiRNA 10 µg/kg50% gene suppression |
AllFacts(Jeffs et 2005 [Reference 2], Belliveau et 2012 [Reference 3]). Belliveau et al al al"Series with variation in siRNA sealing efficiency, uniformity, and repeatability with a diameter of 70 nm or more"Getting StartedFactsHome It is an animal experiment method.
8. Manufacturing process and standards of approved products - read from EPAR
Onpattro(patisiran)FDA approved on August 10, 2018Contact UsFactsHome Our public review report includes preparation and quality control.
"LNP is formed by mixing with aqueous solution containing siRNA (patisiran active ingredient) and an ethanol solution containing four types of lipids."Facts
Manufacturing process of preparation5 major processesCloseFacts1 Preparation of active ingredients and lipid solutions, mixing two solutions to form LNP, diluting to the final concentration of 3%, extra filtration, buffer liquid exchange, primary concentrate, and filling into bioverden filtration, 5-Step Filtration and vial.
This is how to proceed with development."The effect of using different ionized lipids and the relative ratio of four types of lipids were used at the same time."Facts。
About particle size"The particle size of the preparation defines the parameters of the drug delivery. Therefore, it is managed during manufacturing through the LNP forming process.HOME "The particle size of the formulation was chosen to allow the delivery to the liver mainly"Contact UsFactsHome Particle sizeApprox. 60-100 nmContact UsFacts。
Standards during shipping and valid periods include:Facts。
Identification (IPRP-HPLC MS)/Identification (AX-HPLC UV)/Purity and impurities/Containment (AX-HPLC UV)/Identification of lipids and lipid content (RP-HPLC ELSD)／E)tion Rate of Two Chains (siRNA) (Fluorescent Assay)/pH/penetration pressure/Particle size (DLS)ICP-MSResidual ethanol (GC)/Residual EDTA / Insoluble Particles / Endotoxin / Aseptic / Containment Uniformity / Container Contents Volume / Container Obstruction Completeness /siRNA in vitro emission (speed theory t assay)
The report also records discussions on how to determine standard values. Standard width by statistical method originally proposedNot Accepted by CHMP、 "±3SD range or narrower than that"required to propose standards and resultsSome items have narrowed standardsFacts。
Another interesting article is the material 。ian.White film may occur in the vial after transportationThis is a problem. This is the report"Inherent nature of LNP as a reaction to mechanical stress"and AttachmentDo not suck up with a filtered needle, shake or stir the vial, add it to the infusion bag, and mix it gentlyis indicatedFacts。
9. Perspective of Material Technicians 2: Standard items are required specifications of materials as they are
If you draw the underline of the standard item of Chapter 8,Capacity to move in the process(This article explains)
- Lipid content (4 components):Is the blending ratio protected? As seen in Chapter 5, the ratio of the mor directly connects to the activity
- E tion Rate:Is nucleic acid contained in particles?Movement in mixing conditionsFacts
- Particle size (DLS):Report itself“Managed during manufacturing through the LNP formation process”I wroteFacts
- Residual ethanol:It is the back of the process using solvent
- in vitro release:Not only does it include?“Who comes out?”Contact Us According to the report, this is an item added to the standard during the procedureFacts
LNP Even if the composition table is the same,When mixing is different, the particle size and e tion rate will change.Home So standardNot only composition, but also structure and behavior(This article explains) It is the same composition as the requirements of the reposome preparations handled by this series "DDS and Targeting".
There is another important point when looking from the side of measuring lipids. About DLin-MC3-DMA and PEG2000-C-DMG"It is a chemically synthesized lipid compound and is manufactured under acceptable standards from a well-defined starting substance."HOME The information about organic impurities, residual solvents, in impurities (including elemental impurities), and genetic toxic impurities is sufficiently detailed if the material quality characteristics and important process parameters are not defined.CheckFactsHome PEG2000-C-DMG"No animal-derived and human-derived materials used in the manufacturing process"CommentFacts。
Fats used as additives are required to be managed according to the original drugThat is This article explains. In general industrial materials,We need to manage the same impurities profile as the main raw material, for aids and dispersants.Contact Us
10. Path in the body: ApoE escapes only 1 to 2% with endsomes
Why does LNP go to hepatocellular cells? The surface does not contain ligands for the liver. Akinc et al report its mechanismFacts。
"Apoprotein E (apoE), which plays a major role in clearing physiological lipoproteins and intake of liver cells, found working as an endogenous targeted ligand for ionized LNPs. However, I don’t work for [chemical] LNPs.”Facts
This role isIn vitro experiment using recombinant apoEHomeIn vivo experiment with wild mouse and apoE missing mouseSearch Dependence on the receptorLow specific gravity lipoprotein receptor (LDLR) missing mouseConsidered usingFacts。
As an exogenous ligandMultivalent GalNAc CWe also develop methods to bind liver cells to Acyanoprotein receptors (ASGPR), Both endogenous apoE and exogenous GalNAc types look like an effective strategy to deliver ionized LNP to the liverFacts。
What happens after entering cells? Gilleron et al tracks with imaging and electron microscopes.Facts。
"LNP uses cluster-dependent endocytic and macropinocytosis to enter cells in both configurative and inductive pathways depending on cell types."Facts
“By directly detecting colloidal gold particles combined with siRNA, siRNA escapates from endsomes to cell quality only with low efficiency (1–2%), and LNP supposes only within a limited time in a specific compartment with both initial and late endsome characteristics.”Facts
1〜2%The number is changed to the target of material development. Blood retention and intake are already quite efficient. Still2 digits fall out of the last membraneHome If you can improve it, it is a mus。 way that can reduce the dosage.
In fact, Sabnis et al developed a new aminolipid for mRNA is a summary “Mechanical studies have shown that increased efficiency can be attributed to an increase in escaping from endsomes.”FactsHome This paper reports new amino lipids that efficiently deliver mRNA with rodents and primates models, A month of toxicity assessment of toxins and non-human primates did not have any adverse events in the new LNP system.Facts。
Trade-off of "inc」ing reaction" and "keeping stability"but this is also the center. Lipids that are easy to break the membrane are easy to break in the blood.Make a response border with pKaThe design is This contradictionreplace pH difference in the environmentHow to solve this article. This is the same composition as the design of a cut-based linker handled by this series "Antibody Medicine and ADC".
11. Delivering to other than the liver: Efforts in four stages
As seen in Chapter 10, LNP lives in the liverapoE is transported to the receptorHomeIt is necessary to remove from this mechanism in order to deliver it other than the liverThe following are:Research Stage Reportnot approved products.
| Research. | Reported Results | |
|---|---|---|
| Cheng （2020） | SORT (Selective organ targeting)Home Multiple LNPsSubsi "SORT "Systematic design to edit non-liver tissue | SORT lipid nanoparticles targeting lungs, spleen and liverDesign Selectively edits cell species such as epithelial cells, endothelial cells, B cells, T cells, and liver cells. mRNA, Cas9 mRNA, guide RNA, and Cas9 |
| Rurik et （2022） | LNP targeted CD5to T-lymphocytes. Injected into a model of heart failure of the mouse | Delivery to T lymphocytes is observed.CAR-T cells that are transient and effective are generated in the bodyReduced vibration and recovered heart failure after injury. |
AllFacts(Cheng et al 2020 [Reference Material 8], Rurik et al 2022 [Reference Material 9])Both are reports of animal experiments and are not approved treatments.CAR-T cells themselves are handled in this series "CAR-T cells".
12. Issues and Undetermined
(1) Delivery to other than the liver is the stage of research
Chapter 11 reports are animal experiments. LNP preparations targeting organs other than the liver were approved by the FDA or liver as of September 2026.Undetermined / Future。
(2) The mixing method of approved products cannot be specified.
Recent reports"LNP is formed by mixing aqueous and ethanol solutions"CommentFacts、 The condition of the concrete mixing system (T-shaped mixing, micro flow path, etc.), flow, temperature, and lipid concentration was not included in the published materials referred to in this article. Report itself also includes important process parameters and tolerances"Defined"It is said that.
(3) How to increase the efficiency of end-som escape
Gilleron et al. 1 to 2%Estimate in specific cell systems and conditionsHomeFacts。 The primary information that indicates how far this was improved was not confirmed in the scope of the survey.Undetermined / Future。
- LNP is a particle of four components, not a hollow bag.The cryo-TEM image is a s、ical particle with a high electronic density core.Facts
- Ionized lipid pKa determines the effect.The optimal range was 6.2 to 6.5 in 56 comparisons, and DLin-MC3-DMA was 6.44.Facts
- Molar ratio from 40/10/40/10 to 50/10/38.5/1.5.Approval matches with 49.3/10.2/39.0/1.5 even if reversed from the composition of the approved product.The calculation of this paper
- PEG fat is the prerequisite part of "outing".The chain length changes the withdrawal speed and 1.5 % is threshold.Facts
- Production is centered on the process of mixing two liquids. Standards are not only for composition, but also for particle size, sealing rate, and release.Facts
- The most difficult place is escape from the endsome, and the efficiency is 1 to 2%.Facts
13. Glossary
- LNP
- Nanoparticles that are made from four types of lipids, and carry nucleic acids into cells.
- Ionized Lipids
- It is acidic and positively charged, and there is no charge in the vicinity of neutral. Main component of LNP.
- pKa
- pH that goes half. In ionized lipids, it refers to the value of the observation measured by the TNS method.
- TNS Method
- 2-(p-toluidino)-6-Naphthalene sulfonic acid。ce, the method to examine the positive charge of the membrane by pH titration.
- DLin-MC3-DMA
- Ionized lipids used in Onpattro. weight 642.09, reported as pKa 6.44.
- DSPC
- Distealroylphosphatidylcholine. Structural lipids supporting particle structure.
- PEG Fat
- Lipids combined with poly l. Controls particle formation and gradually releases in the body.
- Molar ratio
- ratio of the substance amount of the component. LNP is represented in 50/10/38.5/1.5.
- Contact Us
- The proportion of the added nucleic acid in the particle. Items of shipping standard.
- Multi-Dispersion (PDI)
- Indicator of particle size variation. Smaller particle size.
- Micro flow channel mixing
- How to mix two liquids in milliseconds with a fine flow path.
- Ultrafiltration
- Except solvents and small s in the membrane, the operation to replace or concentrate the buffer liquid.
- apoE
- Apolipoprotein E mediates the ionization of LNP in the blood and facilitates the uptake by liver cells.
- End Som Escape
- Nucleic acids come out of the imported particles. Efficiency is reported to 1 to 2%.
- cryo-TEM
- How to observe frozen samples with a transparent electron microscope. Check the internal structure of the particles.
- New additives
- Additives that are not used in ready-made formulations. More materials are required for examination.
- SORT
- Selective organ targeting. A method of study that changes the target organs by adding supplemental lipids.
14. Reference Materials
- Jayaraman「Maximizing the potency of siRNA lipid nanoparticles for hepatic gene silencing in vivo」Angewandte Chemie International Edition 51(34), 8529–8533 (2012). doi:10.1002/anie.201203263 — pmc.ncbi.nlm.nih.gov
- Jeffs, L.B.「A scalable, extrusion-free method for efficient liposomal encapsulation of plasmid DNA」Pharmaceutical Research 22(3), 362–372 (2005). doi:10.1007/s11095-004-1873-z — pubmed.ncbi.nlm.nih.gov
- Belliveau「Microfluidic Synthesis of Highly Potent Limit-size Lipid Nanoparticles for In Vivo Delivery of siRNA」Molecular Therapy — Nucleic Acids 1, e37 (2012). doi:10.1038/mtna.2012.28 — pmc.ncbi.nlm.nih.gov
- Akinc「Targeted delivery of RNAi therapeutics with endogenous and exogenous ligand-based mechanisms」Molecular Therapy 18(7), 1357–1364 (2010). doi:10.1038/mt.2010.85 — pmc.ncbi.nlm.nih.gov
- Gilleron「Image-based analysis of lipid nanoparticle-mediated siRNA delivery, intracellular trafficking and endosomal escape」Nature Biotechnology 31(7), 638–646 (2013). doi:10.1038/nbt.2612 — pubmed.ncbi.nlm.nih.gov
- FDA／NLM DailyMedONPATTRO (patisiran) Attachment — dailymed.nlm.nih.gov
- European Medicines Agency (CHMP)「Onpattro — Assessment report」EMA/554262/2018（PDF） — ema.europa.eu
- Cheng「Selective organ targeting (SORT) nanoparticles for tissue-specific mRNA delivery and CRISPR-Cas gene editing」Nature Nanotechnology 15(4), 313–320 (2020). doi:10.1038/s41565-020-0669-6 — pmc.ncbi.nlm.nih.gov
- Rurik, J.G.「CAR T cells produced in vivo to treat cardiac injury」Science 375(6576), 91–96 (2022). doi:10.1126/science.abm0594 — pubmed.ncbi.nlm.nih.gov
- Mui, B.L.「Influence of Polyethylene Glycol Lipid Desorption Rates on Pharmacokinetics and Pharmacodynamics of siRNA Lipid Nanoparticles」Molecular Therapy — Nucleic Acids 2, e139 (2013). doi:10.1038/mtna.2013.66 — pmc.ncbi.nlm.nih.gov
- Sabnis「A Novel Amino Lipid Series for mRNA Delivery: Improved Endosomal Escape and Sustained Pharmacology and Safety in Non-human Primates」Molecular Therapy 26(6), 1509–1519 (2018). doi:10.1016/j.ymthe.2018.03.010 — pmc.ncbi.nlm.nih.gov
- NIH／NCBI PubChem"Cholesterol (CID 5997)" — formula and 」 weight — pubchem.ncbi.nlm.nih.gov
- NIH／NCBI PubChem1,2-Distearoyl-sn-phosphero-3-phosphocholine (CID 94190) pubchem.ncbi.nlm.nih.gov
- FDA Drugs@FDANDA 210922 ONPATTRO Approval History — accessdata.fda.gov
15. Response Table of Claim and Sources (Audit)
| Content | Home | |
|---|---|---|
| Compared to 56 types of aminolipids, there was a strong correlation between lipid pKa and mouse FVII gene suppression (ED50), and the optimal pKa range was 6.2–6.5. The pKa of DLin-MC3-DMA (lipid 16) is 6.44, and the ED50 is 0.03 mg/kg. When the pKa is shifted to the acidic side or base, the activity is greatly reduced. TNS (2-(p-toluidino)-6-naphthalene sulfonic acid) is a method to measure pH by dripping up to 0.5 minutes until pH 3-10, and it does not occur when it is free. Changing the distance between esters and amines from 1 to 5 m can change pKa from 4.17 to 7.16. Comparative conditions (up to 3 times in 80 nm membranes, particle size 70-90 nm, multi dispersion 0.11±0.04, the siRNA/total lipid weight ratio is about 56, the optimized mal ratio is 50/10/38.5/1.5, the improvement of ED50 is 0.005 mg/kg and 6 times in the mouse FVII model was obtained, and the same lipid composition was administered to the canines). The standard composition mal ratio is 40/10/40/10, and the optimized mal ratio is 50/10/38.5/1.5, the improvement of ED50 is 0.005 mg/kg and 6 times in the mouse FVII model was obtained, and the same lipid composition was administered to the canines. | Jayaraman et al 2012Source 1 https://pmc.ncbi.nlm.nih.gov/articles/PMC3470698/ | Facts |
| It is a method that can combine the flow of DNA buffer and ethanol-lipid in the T-shaped mixing chamber and expand production without using extrusion. A single-dispersed follicle with less than 200 nm particle size was greater than 80% DNA e efficiency. A single layer, two-layer and multi-layered follicle was mixed with cryo-TEM | Jeffs et al 2005Source 2 https://pubmed.ncbi.nlm.nih.gov/15835741/ | Facts |
| In conventional macroscopic mixing method, it becomes a system with a variation in e efficiency, uniformity, and repeatability with a diameter of 70 nm or more. Micro flow channel mixing enables millisecond mixing at nanoliter scale. The LNP limit size of 20 nm or more has been reproduced in almost complete e tion rate and multi-dispersion at least 0.02. Achieved 50% gene suppression with siRNA 10 μg/kg in mouse liver cells | Belliveau et al 2012Source 3 https://pmc.ncbi.nlm.nih.gov/articles/PMC3442367/ | Facts |
| ApoE acts as an endogenous targeting ligand for ionized LNP and does not work on ionized LNP. In vitro experiments using recombinant ApoE, in vivo experiments with wild and ApoE-deficient mice, and LDLR-deficient mice. Developed a way to combine multivalent GalNAc clusters into ASGPR as exogenous ligands, both endogenous ApoE and exogenous GalNAc types look like a valid strategy for delivery to the liver. | Akinc et al (2010)Source 4 https://pmc.ncbi.nlm.nih.gov/articles/PMC2911264/ | Facts |
| LNP uses cell type-dependent endocytosis and macropinocytosis to enter cells through both configurative and endocytic pathways depending on the cell type. From the direct detection of colloidal gold-labeled siRNA, the escape from endosomes to cell quality is only possible at a low rate of 1 to 2%, and it is quoted only within a limited time that remains in a specific compartment with both initial and late endosome features. | Gilleron et al (2013)Source 5 https://pubmed.ncbi.nlm.nih.gov/23792630/ | Facts |
| ONPATTRO Composition per mL (patisiran 2 mg=patisiran sodium 2.1 mg equivalent, cholesterol 6.2 mg, DLin-MC3-DMA 13.0 mg, DSPC 3.3 mg, PEG2000-C-DMG 1.6 mg, phosphate dihydrogen potassium 0.2 mg, sodium 8.8 mg, hydrogen phosphate dihydrochloride 2.3 mg, water for injection), pH is about 7.0. The formula of patisiran sodium is C412H480N148Na40O290P40, and the weight is 14,304 Da. Prepared as a lipid complex for delivery to hepatocellular cells. | ONPATTROSource 6 https://dailymed.nlm.nih.gov/dailymed/drugInfo.cfm?setid=e87ec36f-b4b4-49d4-aea4-d4ffb09b0970 | Facts |
| The lipid additive helps protect siRNA from degradation in the circulatory system. DLin-MC3-DMA and PEG2000-C-DMG are new additives. DLin-MC3-DMA has been extensively reviewed for related phospholipids. The distribution of the circulatory system is measured to be neutral at pH about 7.4, and the active component is released by endosomes at pH about 5. DLin-MC3-DMA has a molecular formula of C43H79NO2, molecular weight 642.09, two linoleic chains, colorless to light yellow liquids, and rarely melts. PEG2000-C-DMG has a molecular formula of C47H83O2, molecular weight 2,650±300 Da, white to ash white powder, and rarely melts. The chain length of PEG lipids changes during circulation, and the particle without PEG is selected to enter liver cells. DSPC optimizes stability and emulsion with structured lipids, and cholesterol avoids membrane permeability with structured lipids. It indicates that the cryo-TEM image is a spherical particle with a high electro-density core. Particle diameter is approximately 60-100 nm. The particle size of the formulation is controlled during drug production to enable delivery to the liver. LNP was formed by mixing aqueous solution and ethanol solution, and iterative efforts were used to examine the relative ratio of different ionized lipids and four lipids simultaneously. The manufacturing process consists of five main steps. The in vitro release test of siRNA has been added to the standard, and the standard width is less than ±3SD. Both lipids are chemically synthesized and manufactured according to a standard that can be accepted from a well-defined starting substance, and important quality characteristics and important process parameters are defined, as well as detailed impurity information. The white film after transportation is the characteristic of LNP as a response to mechanical stress, inhalation with a filtered needle, and quiet fall-off. | Onpattro, /554262/2018Source 7 https://www.ema.europa.eu/en/documents/assessment-report/onpattro-epar-public-assessment-report_en.pdf | Facts |
| SORT (organ-specific targeting) is a method of systematically adding multiple LNPs to edit non-liver tissues by the addition of a supplementary SORT molecule. SORT-lipid nanoparticles targeting lungs, spleen, and liver were designed to selectively edit epithelial cells, endothelial cells, B cells, T cells, and liver cells. They can be combined with mRNA, Cas9 mRNA, guide RNA, and Cas9/basenuclear protein complex. | Cheng et al (2020)Source 8 https://pmc.ncbi.nlm.nih.gov/articles/PMC7735425/ | Facts |
| A qualified mRNA that codes CAR with LNP targeted CD5 has been delivered to T-lymphocytes, and the effective CAR-T cells generated in the body of the mouse's cardio model have been reduced viability and have recovered heart failure after injury. | Rurik et al (2022) abstractSource 9 https://pubmed.ncbi.nlm.nih.gov/34990237/ | Facts |
| In vivo measured the speed of transfer from LNP to plasma lipoprotein for PEG lipids with a dialkylation chain of 14, 16, and 18 carbons. 1.5% is a threshold concentration that minimizes the influence of the chain length, but still changes the drug content and the distribution in the body. In the C18 chain, the gene suppression of hepatocellular cells is greatly impaired, and the C14 chain has little effect. PEG lipids need to control self-organization during particle formation, while negatively affecting delivery to liver cells and gene suppression. | Mui et al (2013)Source10 https://pmc.ncbi.nlm.nih.gov/articles/PMC3894582/ | Facts |
| The new aminolipid system for mRNA delivery provided efficient delivery and optimal drug delivery with rodent and primate models. One month of toxicity assessment of the drug and non-human primates did not have any adverse events. Improving efficiency can lead to increased escaping from the endosome. | Sabnis et al (2018)Source11 https://pmc.ncbi.nlm.nih.gov/articles/PMC5986714/ | Facts |
| formula of cholesterol C27H46O · weight 386.7 | PubChem CID 5997Source12 https://pubchem.ncbi.nlm.nih.gov/compound/5997 | Facts |
| Formula C44H88NO8P for DSPC (1,2-Diesterol-sn-gly -3-phosphocolin) 79 weight 790.1 | PubChem CID 94190Reference13 https://pubchem.ncbi.nlm.nih.gov/compound/94190 | Facts |
| ONPATTRO (NDA 210922) was approved by the FDA on August 10, 2018 | Drugs@FDAReference14 https://www.accessdata.fda.gov/scripts/cder/daf/index.cfm?event=overview.process&ApplNo=210922 | Facts |
| The molar ratio (MC3 20.2 μg, cholesterol 16.0 μg, DSPC 4.18 μg, PEG lipid 0.60 μg) and molar fraction (49.3/10.2/39.0/1.5). This is almost consistent with 50/10/38.5/1.5 of the paper. Total lipid 24.1 mg, weight ratio of lipid to siRNA about 12 times, weight ratio of siRNA to total lipid 0.083, siRNA 0.140 μg, siRNA about 145 molecules per, ratio of amine to phosphate about 3.6. Molar % when PEG lipid weight is 2,350/2,950 (1.7%/1.4%). The mass of the attachment is divided by the weight of each component. PEG lipid weight has a width of ±300 Da. The ratio of amine/phosphate is the upper limit when one amine is assumed to be electrically charged, and the actual dielectricity is determined by pH and pKa. Not to indicate the manufacturing yield ratio. | The calculation of this article. The mass of the attachment is divided by the weight of each component. PEG lipid weight has a width of ±300 Da. The ratio of amine/phosphate is the upper limit when one amine is assumed to be electrically charged, and the actual dielectricity is determined by pH and pKa. Not to indicate the manufacturing yield ratio. | The calculation of this paper |
| Approval status of LNP preparation targeting organs other than the liver. From 1 to 2 % of end-some escape efficiency | The primary information indicating approval and arrival was not confirmed at the time of the investigation (September 2026). | Undetermined / Future |
| Specific conditions such as mixing method, flow rate, temperature, and lipid concentration used in manufacturing ONPATTRO | The recent public review report says "The LNP is formed by mixing aqueous and ethanol solutions", because the publication referenced in this article did not confirm the description of specific conditions. | |
| Read that pKa is a design problem rather than mixing. The TNS method is used to measure the acidity of the sintered Zeta potential and resin. The PEG lipids were sorted as “additives that are essential in the process, but when used”, and the removal process was embedded in the structure. Read that lipids of additives are required to be managed according to the original drug. ADC’s cut-off linker has been arranged to break the membrane. Delivery to other than the liver into two ways. | and commentary of this article based on the publication content. It is not the opinion of authors and regulatory authorities of each paper | |
| Evaluation of efficacy, effect, clinical results and safety of products | This article is an explanation of material technology and manufacturing technology, and does not evaluate the effects and safety of treatment and vac。s. For approved products, only the composition, manufacturing and quality descriptions listed in the regulators’ publications | |
| Fig. 1 - Fig. 4, Fig. 6, Fig. 7 must be a drawing for explanation, not actual images, particles, or equipment. Hero image and figure 5 are AI-generated images. | Notes by this article |
Last Updated: September 26, 2026 / Source is limited to primary information (FDA approved documentation, recent public review report, Drugs@FDA, PubChem, and reader review paper). They are distinguished from the facts that have been sourced as "instructions" because they contain readings in material and process design. The relationship between pKa and active, endosom escape efficiency, SORT and CD5 target LNP results are both animal and cell experiments, not human. The approval status of LNP preparation targeted for mixing method, flow rate, temperature, etc. of approved products, and the status of approval of LNP preparation targeted for non-liver is not stated because it was not confirmed by the public primary information. This article explains materials technology and manufacturing technology.It does not evaluate the effects and safety of treatments or vac s, and is not medical advice. All diagrams are illustrations for explanation. Figure 1 - Figure 4 · Figure 6 · Figure 7 is a vector drawing, is an image and figure 5 is an AI generation image.These are not actual images, particles, or devices.