Synthesis of aptamer-PEG2000-DSPE.
- Dissolve the lipids of DSPE-PEG2000-Mal in chloroform.
- Dry into a thin film and hydrate with 20 mM HEPES buffer (pH 6.5).
- Meanwhile, activate 3’ thiol-modified aptamer in 100 mM Tris-(2-carboxyethyl) phosphine (TCEP) solution at 4 °C for 30 min.
- Then, add the freshly prepared aptamers to DSPE-PEG2000-Mal solution at a lipid/aptamer molar ratio of 5:1.
- Perform the coupling reaction overnight at 4 °C with gentle stirring.
- Evaluate the conjugation by polyacrylamide gel electrophoresis (PAGE).
- Purify by ultracentrifugation (10,000 g, 15 °C, 15 min) in centrifugal filter tubes (MWCO 10,000).
Preparation of LNPs.
The LNPs were prepared by spontaneous vesicle formation.
- Dissolve approximately 5 mg of lipids, including Dlin-KC2-DMA, DPPC, cholesterol and C16 Ceramide-PEG2000, in 350 µl of ethanol at a molar ratio of 48:10:38:4.
- Slowly add the lipids to 650 µl of 0.62 mg ml–1 siRNA solution (50 mM citrate buffer at pH 4) under a strong vortex.
- Perform dialysis in PBS (155 mM NaCl, 3 mM Na2HPO4 and 1 mM KH2PO4 at pH 7.4) through a regenerated cellulose tubular membrane (MWCO 10,000) for 5 h to remove the ethanol. Increase the external pH by exchanging the buffer at intervals.
Post-insertion of aptamer into the surface of LNPs and purification.
- Add 4 mol% aptamer-PEG2000-DSPE (relative to the total lipids) to the LNPs suspension and incubate in a water bath at 37 °C overnight.
- After incubation, purify the aptamer-LNPs by size exclusion chromatography on a Sepharose CL-4B column using HBS (pH 7.4) as a running buffer to remove external siRNA, unconjugated micelles and chemical reagents.