DMG-PEG 2000

Cell lines

Human hepatocellular carcinoma cell line Hep3B

Preparation Method

TT2-TT8 were formulated with the helper lipid (1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE)), cholesterol, 1,2-dimyristoyl-sn-glycerol, methoxypolyethylene glycol (DMG-PEG2000) (molar ratio 50/10/38.5/1.5 or based on the orthogonal design table), and FLuc mRNA via pipetting for in vitro studies or via a microfluidic based mixing device for in vivo studies.

Reaction Conditions

Hep3B cells were seeded (2 × 10⁴cells per well) into each well of white 96-well plates in 150 µL of culture medium, allowed to attach overnight in growth medium, and transfected by addition of 20 µL of FLuc mRNA-loaded TT LLNs to growth medium. Transfections were performed in triplicate. After 6 h of transfection, culture medium containing TT LLNs was carefully removed, and 50 µL of serum-free EMEM and 50 µL of Bright-Glo luciferase substrate were mixed and added to each well.

Applications

TT2-TT8 LLNs showed minimal to moderate inhibitory effects on Hep3B cells, and showed a significant positive correlation between transfection efficiency and entrapment efficiency.

Animal models

C57BL/6 mice

Preparation Method

C57BL/6 mice were administered intravenously via tail vein injection with free hFIX mRNA, O-TT3 FLuc (an irrelevant mRNA-loaded LLNs as a control), or O-TT3 hFIX at the indicated dosage. Six hours post administration, blood samples were collected and mixed with an anticoagulant solution (3.2% sodium citrate anticoagulant containing 0.17 mg/mL of corn trypsin inhibitor) in a ratio of 9:1, which was then centrifuged for 15 min at 2500 g. hFIX protein level was measured by enzyme-linked immunosorbent (ELISA) assay.

Dosage form

0.55 mg/kg or 1.1 mg/kg

Applications

Wild-type mice produced 1020 ng/mL hFIX at a dose of 0.55 mg/kg and 2057 ng/mL at a dose of 1.1 mg/kg, respectively. No hFIX was detected in the plasma of mice injected with untreated, free hFIX mRNA-, or TT3 LLNs-treated groups

References:

[1]: Li B, Luo X, Deng B, et al. An orthogonal array optimization of lipid-like nanoparticles for mRNA delivery in vivo[J]. Nano letters, 2015, 15(12): 8099-8107.