Chemical Modification Nanotechnology Molecular Biology

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Authors: Heng Wu, Chao Liang, Baosheng Guo, Lingqiang Zhang, Aiping Lu & Ge Zhang

Abstract

Briefly, lipid nanoparticles were prepared by spontaneous vesicle formation after a lipid-ethanol solution was slowly injected into siRNA buffer solution and then dialyzed. The 3’ thiol-modified aptamers were first activated and then conjugated to DSPE-PEG2000-Mal to form aptamer-PEG2000-DSPE. Finally, aptamer-PEG2000-DSPE in the form of micelles was inserted into the surface of the LNPs.

Procedure

Synthesis of aptamer-PEG2000-DSPE.

  1. Dissolve the lipids of DSPE-PEG2000-Mal in chloroform.
  2. Dry into a thin film and hydrate with 20 mM HEPES buffer (pH 6.5).
  3. Meanwhile, activate 3’ thiol-modified aptamer in 100 mM Tris-(2-carboxyethyl) phosphine (TCEP) solution at 4 °C for 30 min.
  4. Then, add the freshly prepared aptamers to DSPE-PEG2000-Mal solution at a lipid/aptamer molar ratio of 5:1.
  5. Perform the coupling reaction overnight at 4 °C with gentle stirring.
  6. Evaluate the conjugation by polyacrylamide gel electrophoresis (PAGE).
  7. 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.

  1. 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.
  2. 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.
  3. 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.

  1. 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.
  2. 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.

Author information

Heng Wu, Chao Liang, Baosheng Guo & Aiping Lu, Institute for Advancing Translational Medicine in Bone & Joint Diseases, Hong Kong Baptist University, Hong Kong SAR, China

Lingqiang Zhang, State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, Beijing, China

Ge Zhang, Institute for Advancing Translational Medicine in Bone & Joint Diseases, Hong Kong Baptist University

Correspondence to: Lingqiang Zhang ([email protected]), Aiping Lu ([email protected]), Ge Zhang ([email protected])

Source: Protocol Exchange (2014) doi:10.1038/protex.2014.053. Originally published online 16 December 2014.

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