The preparation of mRNA-LNPs exploits the self-assembly properties of LNP: negatively charged nucleic acids form electrostatic complexes with positively charged lipids, which then grow through hydrophobic interactions and van der Waals forces among the lipid components. LNPs can be prepared using various methods, such as lipid vesicle extrusion, lipid membrane rehydration, nanoprecipitation, and microfluidic mixing, with rapid mixing of aqueous and lipid solutions being the most commonly employed. After preparation, mRNA-LNPs are characterized for parameters such as size, charge, and encapsulation efficiency using multiple techniques. Fluorescent labeling may also be employed to track intracellular uptake and verify the presence of encapsulated mRNA. The preparation and characterization process is shown in the figure below:

All preparation protocols provided by our company are adapted from published Nature references^[9], and we do not guarantee their practical application outcomes. Lipid ratios may be adjusted according to experimental goals and conditions.

Lipid Solutions:
1. Weigh 15 mg of DLin-MC3-DMA (MC3) using a balance, then add 200 μL of absolute ethanol to dissolve it, resulting in a concentration of 75 mg/mL.
2. Weigh 10 mg of DSPC using a balance, then add 1.0 mL of absolute ethanol to dissolve it, with a final concentration of 10 mg/mL.
3. Weigh 10 mg of cholesterol using a balance, add 1.0 mL of absolute ethanol to dissolve it, achieving a final concentration of 10 mg/mL.
4. Weigh 10 mg of DMG-PEG on a balance, and then dissolve it in 1.0 mL of pure ethanol to obtain a final concentration of 10 mg/mL.

Mixed Lipid Solution (Solution I):
Mix 13.3 μL of DLin-MC3-DMA solution, 24.6 μL of DSPC solution, 46.4 μL of cholesterol solution, and 11.7 μL of DMG-PEG solution thoroughly to obtain a clear mixed solution I. Mixed solution I contains 19 μg of total lipids per μL of absolute ethanol.

mRNA Payload:
Dissolve mRNA in 10 mM citrate buffer (10 mM, pH 4). The lipid mixed solution obtained in the above steps and the aqueous buffer containing mRNA are rapidly mixed to achieve a final weight ratio of 40/1 (total lipid/mRNA, wt/wt).
Note: The proportion of mRNA can be increased to reduce potential lipid toxicity.

Mixing Methods:
Three commonly used methods facilitate rapid mixing of the lipid solution with the mRNA solution: pipette mixing (for small-scale preparation), vortex mixing (for medium-scale preparation), and microfluidic mixing (for large-scale preparation).

A) Pipette Mixing (Small-Scale Preparation)
1. Add 16.8 μL of the above-obtained mixed solution I into a RNase-free 1.5 mL tube.
2. Add 1.2 μL of ethanol to the tube and mix thoroughly to obtain mixed solution II.
3. Take another RNase-free 1.5 mL tube, add 46 μL of citrate buffer (10 mM, pH 4), then add 8 μL of mRNA (1.0 mg/mL) to the tube and mix well.
4. Add 54 μL of mRNA buffer solution to mixed solution II obtained in step 2, followed immediately by rapid pipetting up and down for 20–30 s (manual mixing).
Note: The volume ratio of water to ethanol is 3:1. Mix by pipetting immediately after combining; otherwise, the activity of the mRNA-LNP mixture may be affected.
5. Incubate the solution obtained in step 4 at room temperature for 15 min.
6. Dialyze the above solution in 1× PBS using a dialysis tube (MWCO 3500) for more than 1 h to remove ethanol and acidic buffer.
7. After dialysis, transfer the solution to a RNase-free 1.5 mL tube and measure the volume.
8. Add 1× PBS solution to bring the total volume to 800 μL.
Note: The resulting solution can be stored short-term at 4°C.

B) Vortex Mixing (Medium-Scale Preparation)
1. Add 21 μL of the above-obtained mixed solution I into a RNase-free 1.5 mL tube.
2. Add 9 μL of ethanol to the tube and mix thoroughly to obtain mixed solution II.
3. Take another RNase-free 1.5 mL tube, add 80 μL of citrate buffer (10 mM, pH 4), then add 10 μL of mRNA (1.0 mg/mL) to the tube and mix well.
4. Set the vortex mixer to "ON" with the speed level at "1".
5. Vortex the mRNA buffer solution at medium speed on the vortex mixer, then quickly transfer 30 μL of mixed solution II into the vortexing solution, and vortex the resulting mixture for 20–30 s.
Note: The volume ratio of water to ethanol is 3:1. Mix immediately after combining; otherwise, the activity of the mRNA-LNP mixture may be affected.
6. Incubate the solution obtained in step 5 at room temperature for 15 min.
7. Dialyze the above solution in 1× PBS using a dialysis tube (MWCO 3500) for more than 1 h to remove ethanol and acidic buffer.
8. After dialysis, transfer the solution to a RNase-free 1.5 mL tube and measure the volume.
9. Add 1× PBS solution to bring the total volume to 1000 μL.
Note: The resulting solution can be stored short-term at 4°C.

C) Microfluidic Mixing (Large-Scale Preparation)
The specific operational protocol is highly instrument-dependent. It is recommended to refer to the official website instructions of the specific instrument used.

To ensure LNP quality and performance, key parameters such as particle size, polydispersity index (PDI), surface charge, and RNA encapsulation efficiency are routinely characterized.