This protocol is for reference only. The actual application conditions may need to be optimized according to your specific experimental needs.
Preparation of Glu-urea-Lys ligand-coupled lipid nanoparticle/siRNA system [1-2]:
1.Reagent preparation: DSPE-PEG2000-NH₂, N-hydroxysuccinimide (NHS), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), borate buffer (0.1M, pH 8.5), dialysis bag (MWCO 3.5kDa), siRNA, citric acid buffer (10mM, pH 4.0),
2. Preparation of Glu-urea-Lys-DSPE-PEG2000 conjugate
2.1 Carboxyl activation: Dissolve Glu-urea-Lys (1eq), NHS (2eq), and EDC (2eq) in DMF and stir at room temperature for 4h.
2.2 DSPE-PEG2000 coupling: The activated product was added dropwise to DSPE-PEG2000-NH₂ (1.2eq) in borate buffer, pH 8.5, and reacted at room temperature in the dark for 24h.
2.3 Purification: The reaction mixture was placed in a dialysis bag, sealed and immersed in PBS buffer (4°C, in the dark, 48h) to remove small molecule impurities, and the conjugate was obtained after freeze-drying.
2.4 Verification: MALDI-TOF MS was used to verify the molecular weight of the compound (expected molecular weight: DSPE-PEG2000 + Glu-urea-Lys ≈ 2800Da + 348Da).
3. Preparation of lipid nanoparticles (LNP) and siRNA loading
3.1 Preparation of lipid solution: Each lipid was dissolved in ethanol (total lipid concentration 10mg/mL) according to the ratio of DLin-MC3-DMA (50%), cholesterol (38.5%), DSPC (10%), and Glu-urea-Lys-DSPE-PEG (1.5%), and ultrasonicated in a 60°C water bath until completely dissolved.
3.2 Preparation of aqueous phase: siRNA was dissolved in citric acid buffer (0.2mg/mL).
3.3 Microfluidic mixing: The lipid phase and the aqueous phase were mixed at a flow rate ratio of 3:1 (total flow rate 12mL/min), and the outlet was collected in PBS (pH 7.4).
3.4 Dialysis purification: The reaction mixture was placed in a dialysis bag, sealed and immersed in PBS buffer (4°C, protected from light, 48h), and the buffer was changed 3 times during the period.
4. Quality control
4.1 Particle size: DLS detection (80-100nm, PDI < 0.2).
4.2 Encapsulation efficiency: RiboGreen method (> 95%).
4.3 siRNA integrity: agarose gel electrophoresis (no free siRNA bands).
Notes
1. Aseptic operation: In vivo experiments require filtration sterilization (0.22μm filter membrane).
2. siRNA stability: LNPs must be stored at -80°C after preparation to avoid repeated freezing and thawing.
3. In vivo distribution: Bioluminescence imaging (such as DiR-labeled LNPs) can be supplemented to observe targeting.
References:
[1]. Lee J B, Zhang K, Tam Y Y C, et al. A Glu-urea-Lys ligand-conjugated lipid nanoparticle/siRNA system inhibits androgen receptor expression in vivo[J]. Molecular Therapy Nucleic Acids, 2016, 5.
[2]. Zhou W, Huang J, Xiao Q, et al. Glu-Urea-Lys Scaffold Functionalized Superparamagnetic Iron Oxide Nanoparticles Targeting PSMA for In Vivo Molecular MRI of Prostate Cancer[J]. Pharmaceutics, 2022, 14(10): 2051.
