Preparation method of SM-102 for lipid nanoparticles (LNPs)[1-2]
This experimental protocol is compiled based on research content and is for reference only. Please adjust according to actual needs.
(1) Reagent preparation: Dissolve SM-102 in ethanol to prepare a 10mg/mL stock solution. Dissolve cholesterol (Catalog No. GC17398), DSPC (Catalog No. GC41832), and DMG-PEG 2000 (Catalog No. GC39784) separately in anhydrous ethanol. Dissolve mRNA in 50mM sodium citrate buffer (pH=4.0). If LNPs need to be labeled, add the DiD fluorescent probe to the lipid ethanol solution at a concentration of 0.2%.
(2) Preparation of lipid nanoparticles: Add 572μL of 10mg/mL SM-102, 240μL of 10mg/mL cholesterol, 127μL of 10mg/mL DSPC, and 61μL of DMG-PEG 2000 per milliliter of lipid mixture. Mix the solution thoroughly to obtain a clear solution. This mixture contains 10mg of total lipids. The lipid composition is SM-102/cholesterol/DSPC/DMG-PEG at a molar ratio of 50:38.5:10:1.5.
(3) Mixing mRNA with LNPs: The weight ratio of RNA to lipid is 0.05 (wt/wt). Rapid mixing can be achieved by three methods: pipette mixing, vortex mixing, and microfluidic mixing.
a. Pipette mixing method: Aspirate 3mL of mRNA solution and quickly add 1mL of lipid mixture solution (generally using a 1:3 ratio of ethanol lipid mixture to aqueous buffer). Rapidly pipette up and down for 20-30 seconds. Incubate the resulting solution at room temperature for up to 15 minutes.
b. Vortex mixing method: Vortex 3mL of mRNA solution at medium speed on a vortex mixer. Quickly add 1mL of lipid mixture solution into the vortexing solution (usually using a 1:3 ratio of ethanol lipid mixture to aqueous buffer). Continue vortexing the resulting dispersion for 20-30 seconds. Incubate the resulting solution at room temperature for up to 15 minutes.
c. Microfluidic mixing method: Mix 3mL of mRNA buffer with 1mL of lipid mixture solution in a microfluidic device at a total flow rate of 12mL/min (generally using a 1:3 ratio of ethanol lipid mixture to aqueous buffer).
(4) Purification of lipid nanoparticles: After mixing, dialyze the LNPs against PBS (pH=7.4) for 2 hours, sterile filter through a 0.2μm filter, and store at 4°C.
Notes:
(1) LNPs prepared by this method have a particle size of approximately 100nm, a polydispersity index of less than 0.2, and an encapsulation efficiency of up to 98.96%.
(2) LNPs maintain stable particle size and PDI during storage at 4°C for 4 weeks, but mRNA content decreases over time.
(3) For safety and health, please wear a lab coat and disposable gloves during operation.
References:
[1] Escalona-Rayo O, Zeng Y, Knol RA, et al. In vitro and in vivo evaluation of clinically-approved ionizable cationic lipids shows divergent results between mRNA transfection and vaccine efficacy. Biomed Pharmacother. 2023 Sep;165:115065.
[2] Naidu GS, Yong SB, Ramishetti S, et al. A Combinatorial Library of Lipid Nanoparticles for Cell Type-Specific mRNA Delivery. Adv Sci (Weinh). 2023 Jul;10(19):e2301929.
