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Protocol: Lipid Nanoparticle (LNP) Preparation

 

Protocol: Lipid Nanoparticle (LNP) Preparation

1. Preparation of Lipid Solution

In a 50 mL centrifuge tube, mix the following lipid stock solutions in ethanol:

Component

Stock Conc.

Volume

Ionizable lipid

5 mM

300 µL

Helper lipid

5 mM

60 µL

Cholesterol

5 mM

231 µL

DMG‑PEG2000

1 mM

45 µL

Total Volume

 

636 µL

2. Preparation of mRNA Solution

Dissolve 40 µg of mRNA in 1.9 mL of 20 mM Malic or Citric Acid Buffer (pH 3.0).

3. LNP Assembly

Quickly add the mRNA solution into the lipid solution while vortexing for 10 s.

Incubate the mixture at room temperature for 10 min.

4. Dilution and Buffer Exchange

Add 30 mL of TBS buffer to the mixture to form the LNP solution.

5. LNP Concentration (1-2 hours)

- Transfer 15 mL of the LNP solution to an Amicon® Ultra‑15 mL 100 kDa ultrafiltration tube.

- Centrifuge at 2,000 × g for 5 min.

- Discard the filtrate. Re‑fill the retentate to 15 mL with LNP solution and mix gently.

- Repeat the centrifugation/resuspension until the entire batch is concentrated to ~500 µL.

6. Final Concentration and Volume Adjustment (0.5-1 hours)

- Transfer the 500 µL concentrate to an Amicon® Ultra‑0.5 mL 100 kDa ultrafiltration tube.

- Continue centrifugation until the volume is <200 µL.

- Combine retentates and bring to 200 µL with TBS.

- Final LNP concentration: 0.2 mg mL⁻¹.

7. Storage

- Store the LNP at 4 °C overnight before use.

- Recommended maximum storage: 1 week.

8. Quality Control (Optional)

- Dilute 1 µL of LNP into 1 mL TBS 

- Measure particle size and zeta potential by DLS or equivalent.

 

by Xuehao Zhang

05/23/2025

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