The LNPs used in this study comprised a lipid mixture consisting of C12-200 (amino lipid, Axolabs), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), DMPE-mPEG2000 (PEG-lipid), and cholesterol at a 52.2:15.6:8.7:23.6 mass ratio, respectively (DOPE, PEG-lipid, and cholesterol from Avanti Polar Lipids). LNPs were prepared by rapid microfluidic mixing (Precision NanoSystems) of mRNA and sgRNA in acetate buffer at pH 4.0 with the lipid mixture suspended in ethanol (3:1 aqueous to organic volume ratio). After mixing, LNPs were diluted and dialyzed into PBS, concentrated as needed using 100k MWCO spin cartridges (Amicon), and 0.2 µm sterile filtered. mRNA production and sequence optimization was performed as previously described32 (link),59 (link). gRNA was procured from Synthego (sRGN Alb-T1), Avecia (SpyCas9 Alb-T1), and Agilent (modified sRGN Alb-T1 constructs). LNPs were characterized by dynamic light scattering (DLS) using a Wyatt Nanostar, Ribogreen (Thermo Fisher Scientific), for endotoxin (Endosafe, Charles River), by UPLC (Thermo Fisher Scientific), and cryoTEM (MIT) assays.
Peg lipid
Manufactured by Avanti Polar Lipids
Sourced in United States
PEG-lipid is a type of lipid compound that incorporates polyethylene glycol (PEG) into its structure. PEG-lipids are used in the formulation of liposomal and other lipid-based drug delivery systems to improve the pharmacokinetic properties of the encapsulated or associated drugs.
Automatically generated - may contain errors
Lab products found in correlation
E coli poly a polymerase, by New England Biolabs (1 mentions)
1 methylpseudouridine 5 triphosphate, by TriLink (1 mentions)
Vaccinia capping system, by New England Biolabs (1 mentions)
Dlin mc3 dma, by MedChemExpress (1 mentions)
Quant it ribogreen rna assay kit, by Thermo Fisher Scientific (1 mentions)
Cholesterol, by Merck Group (1 mentions)
Ribogreen, by Thermo Fisher Scientific (1 mentions)
Endosafe, by Charles River Laboratories (1 mentions)
3 protocols using peg lipid
1
Lipid Nanoparticle Formulation and Characterization
2
Giant Unilamellar Vesicle Preparation
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1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[poly(ethylene glycol)2000-N’-carboxyfluorescein] (DSPE-PEG(2000) CF), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-2000] (DSPE-PEG(2000) Biotin. Hereafter referred to as “PEG lipid” for short) were purchased from Avanti Polar Lipids (Alabaster, USA). Cholesterol was purchased from Nacalai Tesque (Kyoto, Japan). These lipids were stored in a freezer at -20°C to avoid any degradation of lipids and dissolved in chloroform (100mg/mL concentration) prior to experiments. Fluorescent proteins, transferrin from human serum Alexa Fluor 647 conjugate (TA647, for short) and Alexa Fluor 488 Fluorescent Streptavidin Conjugates (Avidin AF488) were purchased from Invitrogen (Carlsbad, USA). Aqueous solution encapsulated in giant vesicles contains 50 mM HEPES-KOH (pH 7.6), 200 mM sucrose, and 1.5 μM TA647. Buffer solution contains 50 mM HEPES-KOH (pH 7.6), 200 mM glucose.
3
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.
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