Lipid 8

Preparation of Lipid 8-Based Lipid Nanoparticles^[1]

This protocol is adapted from research data and provided for reference only. Adjustments may be necessary based on specific experimental requirements.

1. Reagent Preparation: Prepare Lipid 8, distearoylphosphatidylcholine (DSPC), cholesterol, DMG-PEG2000, mRNA to be encapsulated, ethanol solution, and citrate buffer (pH=4.0).

2. Lipid Nanoparticle Preparation: Use a microfluidic mixing device for preparation. Dissolve Lipid 8, DSPC, cholesterol, and DMG-PEG2000 in ethanol at a molar ratio of 40:10:48.5:1.5 to form the lipid phase. Dissolve the mRNA in citrate buffer (pH=4.0) to form the aqueous phase. Mix the lipid phase and the aqueous phase at a volume ratio of 3:1 (lipid to mRNA weight ratio of 27:1), with a total flow rate of 12mL/min. Immediately after mixing, dialyze against PBS (pH=7.4) for 20-24 hours to remove ethanol and form stable mRNA-loaded lipid nanoparticles (LNPs).

5. Imaging and Quantitative Analysis: Image on a single plane using a confocal microscope. The excitation/emission wavelengths for DQ-BSA-RED are 590nm and 620nm, respectively.

Precautions:

(1) The prepared LNPs should have a particle size distribution of 40-80nm, a Zeta potential between -7 and 2mV, and an mRNA encapsulation efficiency greater than 70%. The LNPs can be stably stored for over one month under refrigeration at 2-8°C.

(2) For your safety and health, please wear a lab coat and disposable gloves during operation.

References:
[1] 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.