LNP-siRNA Protocol
LNP-siRNA v2.110-step protocol for LNP-siRNA preparation
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siRNA Reconstitution
Reconstitute lyophilized siRNA in RNase-free water to achieve final concentration of 10 mg/mL. Ensure the reagent is at room temperature before reconstitution.
LNP Components Preparation
Prepare ionizable lipid, DSPC, cholesterol, and PEG-lipid at specified molar ratios. Dissolve each component separately in ethanol before mixing.
Citrate Buffer Preparation
Prepare 20 mM citrate buffer pH 3.0 for downstream dilution. Filter sterilize through 0.22 μm filter.
siRNA-Lipid Mixing
Mix siRNA solution with lipid ethanol solution at 1:3 volume ratio using microfluidic device. Maintain precise flow rate ratio.
Encapsulation Quenching
Dilute the mixture 1:1 with citrate buffer (pH 3.0) to quench the encapsulation process. Perform immediately after mixing.
Buffer Exchange
Dialyze against PBS pH 7.4 to remove ethanol and neutralize pH. Use 10 kDa MWCO dialysis cassette.
Filtration & Sterilization
Filter through 0.22 μm filter under sterile conditions. Test filter integrity after use (bubble point test).
Quality Control Sampling
Collect samples for encapsulation efficiency (RiboGreen) and particle size analysis (NTA). Store at 4°C until analysis.
Final Formulation
Adjust concentration to 1 mg/mL total siRNA using sterile PBS. Aliquot into cryovials with proper labeling.
Storage
Store final product at 2-8°C with continuous temperature monitoring. Do not freeze. Log temperature readings.