Amines, epoxides, acyl chlorides, TEA were purchased from Sigma Aldrich (Burlington, Massachusetts, USA), Tokyo Chemical Industry (TCI, Tokyo, Japan) and Ambeed (Arlington Heights, Illinois, USA). Alanine transaminase (ALT) colorimetric activity assay kit (#700260) and aspartate aminotransferase (AST) colorimetric activity assay kit (#701640) were purchased from Cayman Chemical (Ann Arbor, Michigan, USA). 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG 2000) and cholesterol were obtained from Avanti Polar Lipids (Alabaster, Alabama, USA). DLin-MC3-DMA and SM-102 were purchased from MedChem Express (Monmouth Junction, New Jersey, USA). Cas9 mRNA (5moU) was purchased from TriLink (San Diego, California, USA). Highly modified sgRNA target mouse TTR (guide No. G211) was chemically synthesized by AxoLabs (Kulmbach, Bayern, Germany) based on the previous publication43 (link). Anhydrous DCM, porcine liver esterase, amiloride hydrochloride, chlorpromazine, genistein, methyl-beta-cyclodextrin and TTR siRNAs (#NM_013697, siRNA IDs: SASI_Mm01_00076059, SASI_Mm01_00076060 and SASI_Mm01_00076061) were purchased from Sigma Aldrich (Burlington, Massachusetts, USA).
Dlin mc3 dma
Manufactured by MedChemExpress
Sourced in United States
DLin-MC3-DMA is a lipid formulation component used in the development of lipid nanoparticle (LNP) drug delivery systems. It is a cationic lipid that can be used to facilitate the encapsulation and delivery of nucleic acid-based therapeutics, such as mRNA and siRNA.
Automatically generated - may contain errors
Lab products found in correlation
1 2 dioleoyl sn glycero 3 phosphoethanolamine, by Avanti Polar Lipids (2 mentions)
Cholesterol, by Merck Group (2 mentions)
1 2 distearoyl sn glycero 3 phosphocholine dspc, by Avanti Polar Lipids (2 mentions)
Chlorpromazine, by Merck Group (1 mentions)
Amiloride hydrochloride, by Merck Group (1 mentions)
Epoxides, by Merck Group (1 mentions)
Porcine liver esterase, by Merck Group (1 mentions)
Acyl chlorides, by Merck Group (1 mentions)
Genistein, by Merck Group (1 mentions)
Sm 102, by MedChemExpress (1 mentions)
Methyl β cyclodextrin, by Merck Group (1 mentions)
Amines, by Merck Group (1 mentions)
Anhydrous dcm, by Merck Group (1 mentions)
1 2 distearoyl sn glycero 3 phosphocholine, by Avanti Polar Lipids (1 mentions)
1 2 dimyristoyl rac glycero 3 methoxypolyethylene glycol 2000 dmg peg2000, by Avanti Polar Lipids (1 mentions)
Dmg peg 2000, by MedChemExpress (1 mentions)
Amicon ultra centrifugal filter, by Merck Group (1 mentions)
Cleancap technology, by TriLink (1 mentions)
Jetpei, by Polyplus Transfection (1 mentions)
E coli poly a polymerase, by New England Biolabs (1 mentions)
9 protocols using dlin mc3 dma
1
Lipid Nanoparticle Synthesis and Characterization
2
Lipid-nanoparticle mRNA Encapsulation Protocol
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Lipid-nanoparticle (LNP) formulations were prepared using a previously described method (Lee et al., 2022 (link)). Briefly, lipids were solubilized in ethanol containing cationic lipid (D-Lin-MC3-DMA) (MedChemExpress, NJ, USA), 1, 2-distearoyl-sn-glycero-3-phosphocholine (DSPC) (Avanti Polar Lipids, NY, USA), cholesterol (Sigma, MA, USA) and DMG-PEG 2000 (MedChemExpress, NJ, USA) at molar ratios of 50:10:38.5:1.5. The lipid mixture was combined with 25 mM sodium acetate buffer (pH 4.5) containing mRNA at a ratio of 1:3 using NanoAssemblr® IGNITE NxGen Cartridges (Precision NanoSystems Inc., BC, Canada). LNP-encapsulated mRNA samples were dialyzed against PBS (pH 7.4) at 4 °C and then concentrated using Amicon Ultra Centrifugal Filters (10 K MWCO; Millipore, Burlington, MA, USA) before being passed through 0.45-μm filters.
3
Formulation and Characterization of Lipid Nanoparticles for mRNA Delivery
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The ionizable polyamine-lipid cores, prepared as described above, or DLin-MC3-DMA (MedChem Express, Monmouth Junction, NJ) were combined into an ethanol phase with cholesterol (Sigma-Aldrich), DOPE (Avanti, Alabaster, AL), and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) (C14-PEG2000, Avanti) at molar ratios of 35:46.5:16:2.5, respectively, in a total volume of 112.5 μl. A separate aqueous phase was prepared consisting of 25 μg of luciferase (TriLink BioTechnologies, San Diego, CA) or EPO (TriLink BioTechnologies) mRNA and 10 mM citrate buffer (pH 3) in a total volume of 337.5 μl. All mRNA was N1-methyl-pseudo-U–capped with CleanCap technology offered by TriLink BioTechnologies. The ethanol and aqueous phases were combined through channels in a microfluidic device using a syringe pump as previously described (39 (link)). NPs were dialyzed against PBS for 2 hours before sterile filtration through syringe filters with 0.2-μm pores and stored at 4°C. JetPEI (Polyplus Transfection, New York, NY)–mRNA complexes were prepared according to manufacturer protocols with N/P = 7. All materials were prepared and handled ribonuclease-free throughout the synthesis, formulation, and characterization steps.
4
Chikungunya Virus mRNA-LNP Production
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The production of mRNA-LNP was described previously (Zhang et al., 2020 (link)). Briefly, RNA was produced using T7 RNA polymerase on linearized plasmids (pok12-5′UTR-CHIKV-E2-E1-3′UTR) encoding CHIKV E2-E1 protein. The UTP was substituted with 1 mψ (1-methylpseudouridine-5′-triphosphate) (TriLink BioTechnologies) to improve protein expression. Then the transcriptional RNA was added with Cap1 and poly(A) tails by using the Vaccinia Capping System and E. coli poly(A) polymerase (New England Biolabs), respectively. The mRNA was stored at −80 °C freezer.
For LNP production, D-Lin-MC3-DMA (MedChemExpress), DSPC (Avanti Polar Lipids), cholesterol (Sigma), and PEG-lipid (Avanti Polar Lipids) were solubilized in ethanol respectively and mixed with a molar ratio of 50:10:38.5:1.5. The lipid mixture was then mixed with an aqueous buffer (50 mmol/L citrate buffer [pH 4.0]), reaching the final lipid concentration of 7.37 mg/mL. Then the lipid mixture and mRNA were added into the NanoAssemblr™ with the volume ratio of 1:3 to obtain mRNA-LNP. Then the diluted LNP-encapsulated mRNA samples were concentrated to appropriate volume and passed through 0.22 μm filters. The produced mRNA-LNP was stored at 4 °C until use. The encapsulation efficiency was measured with the Quant-iT RiboGreen RNA Assay Kit (Life Technologies) using a microplate reader.
For LNP production, D-Lin-MC3-DMA (MedChemExpress), DSPC (Avanti Polar Lipids), cholesterol (Sigma), and PEG-lipid (Avanti Polar Lipids) were solubilized in ethanol respectively and mixed with a molar ratio of 50:10:38.5:1.5. The lipid mixture was then mixed with an aqueous buffer (50 mmol/L citrate buffer [pH 4.0]), reaching the final lipid concentration of 7.37 mg/mL. Then the lipid mixture and mRNA were added into the NanoAssemblr™ with the volume ratio of 1:3 to obtain mRNA-LNP. Then the diluted LNP-encapsulated mRNA samples were concentrated to appropriate volume and passed through 0.22 μm filters. The produced mRNA-LNP was stored at 4 °C until use. The encapsulation efficiency was measured with the Quant-iT RiboGreen RNA Assay Kit (Life Technologies) using a microplate reader.
5
Preparation of mRNA-Loaded Lipid Nanoparticles
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mRNA-LNPs were prepared as previously described [20 (link),21 (link)] with some modifications. The aqueous phase was prepared using 50 mM citrate buffer (pH 3.0) and the luciferase mRNA. The ethanol phase was prepared with four lipid components: ionizable lipid DLin-MC3-DMA (MedChemExpress, Monmouth Junction, NJ, USA), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC, Avanti, Alabaster, AL, USA), cholesterol (NACALAI TESQUE, Kyoto, Japan), and DMG-PEG2000 at a molar ratio of 50:10:38.5:1.5. The aqueous and ethanol phases were mixed using a NanoAssemblr Benchtop (Precision NanoSystems Inc., Vancouver, BC, Canada) with a microfluidic device at a 1:3 volume ratio. The mRNA-LNP was transferred immediately to SnakeSkin™ Dialysis Tubing (10,000 MWCO, Thermo Fischer Scientific Inc., Waltham, MA, USA) and dialyzed overnight at 4 °C in PBS (pH7.4) to remove ethanol. The volume of PBS buffer was approximately 500–1000 × the sample fraction volume. After dialysis, the sample was concentrated using Amicon Ultra (100,000 Da, Merck KGaA, Darmstadt, Germany) and filtered through a 0.45 μm membrane filter.
6
Lipid Nanoparticle Formulation Protocol
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The DLin-MC3-DMA was purchased from MedChemExpress (Monmouth Junction, NJ, USA). SM102 was purchased from Hanmi Fine Chemical Co. Ltd. (Siheung, South Korea). 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2‐distearoyl‐sn‐glycero‐3‐phosphocholine (DSPC), and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). Cholesterol and 6,6′-trehalose dibehenate (TDB) were purchased from Sigma-Aldrich (Burlington, Massachusetts, USA). 6,6′-trehalose dioleate (TDO) and alkyl lithocholate were synthesized as reported [29 ]. Other reagents for the synthesis were purchased from Sigma–Aldrich and Tokyo Chemical Industry (Tokyo, Japan) and were used without further purification.
7
Polyamine-Based Lipid Nanoparticle Synthesis
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Core 200 was customized from Enamine (Monmouth Junction, NJ) and other polyamine cores were purchased from Sigma Aldrich, Tokyo Chemical Industry (TCI), and Alfa Aesar. Epoxydodecane (C12), epoxytetradecane (C14), epoxyhexadecane (C16), 4-methoxybenzoic acid, N,N’-Dicyclohexylcarbodiimide (DCC), N-Hydroxysuccinimide (NHS), HSP47 siRNA pool (NM_001111043, NM_001111044, and NM_009825) and Cy5-siRNA were purchased from Sigma Aldrich. GFP siRNA (cat. P-002048-01-50) and DharmaFECT transfection reagents were purchased from Horizon Discovery Ltd. 1% agarose gels with SYBR™ Safe (#A42100) were purchased from ThermoFisher. Recombinant mouse TGF-β1 (cat. 7666-MB) was obtained from R&D. DSPC (#850365), cholesterol (#700100), and C14-PEG2000 (#880150) were bought from Avanti Polar Lipids. DLin-MC3-DMA was purchased from MedChem Express (Monmouth Junction, NJ). Luciferase mRNA was produced through an in vitro transcription (IVT) method46 (link).
8
Formulation of Lipid Nanoparticles for Delivery
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Gelatin GELITA® MedellaPro® < 100, porcine gelatin, 228 g Bloom, pharmaceutical-grade was purchased from GELITA® Deutschland GmbH, Eberbach, Germany. Protamine sulfate was purchased from Sigma-Aldrich, Darmstadt, Germany. JetMessenger (JetM) and JetPrime (JetP) were purchased from Polyplus-transfection®, Illkirch, France. Branched polyethyleneimine (PEI), Mw~25,000, was purchased from Sigma-Aldrich Darmstadt, Germany. Lipofectin was purchased from Invitrogen, Thermo Fisher Scientific, Darmstadt, Germany. Purified water was obtained from a Milli-Q water purification system (Merck Millipore, Darmstadt, Germany), and is referred to as MQ water. DLin-MC3-DMA was purchased from MedChemExpress (Middlesex County, NJ, USA), Cholesterol was purchased from Sigma-Aldrich Darmstadt, Germany, DSPE-PEG 2000 and DPPC were a kind gift from Lipoid GmbH, Ludwigshafen, Germany.
9
Lipid-based Nanoparticle Synthesis
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DLin-MC3-DMA was purchased from MedChem Express (Bio-Connect, Huissen, The Netherlands). CSL3 lipid was kindly provided by Jeanne Leblond Chain (French Institute of Health and Medical Research, Bordeaux, France). 45
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