Fig. 3: Alkyne lipids facilitate the endosomal fusion and release of mRNA from LNPs.

a Schematic of fusion between A6 (blue) or cKK-E12 (red) vesicles and endosomal vesicles by MD simulation. DOPE and DOPC are in green, LBPA is in purple, cholesterol is in gray. b Estimated vesicle merging states between A6 or cKK-E12 and endosomal vesicles. Merging state is defined as the ratio between the neck area and the largest intersection area of the endosomal vesicle (Error bars: variation of the neck area). c Percentile of A6 or cKK-E12 lipid mixed in endosomal vesicles during fusion simulation. d Schematic of lipid restructuring movements in LNPs during fusion. e MD calculations of lipid tail protrusion in cKK-E12 and A6 membranes (n = 1000). Student’s T-test. Panel on the right: a snapshot of A6 lipid protruding out the membrane with one tail. f Lateral diffusion coefficient of A6 and cKK-E12 lipid in singular membranes calculated from the mean square displacement using linear regression (n = 5000) (Error bars: errors between individual lipids). Student’s T-test. Penal on the right: trajectory of A6 or cKK-E12 lipid within the singular membrane over 40 ns simulation. g Free energy profile of A6 and cKK-E12 lipid sprouting and flip-flop action. h, i hemolysis analysis of variable LNPs in acidic and neutral pH conditions (n = 4), Student’s T-test. j Fluorescence resonance energy transfer (FRET) based membrane fusion assay. Rhodamine-PE and NBD-PE dual labeled endosomal vesicles (donor vesicles) were mixed with non-fluorescent labeled cKK-E12, A6, or Syn-3 LNPs. NBD fluorescence was monitored at 540 nm with excitation at 465 nm upon mixing. Mixing of un-labeled vesicles were subtracted as blank. k Time-lapse images of released free mRNA in primary hepatocytes treated with Cy5-mRNA LNPs in 10% serum. Hepatocytes were highlighted in yellow dotted circles. Yellow arrows indicate released free fluorescent mRNA observed within cytoplasm. Quantifications presented on right were based on the fluorescence intensity around the straight dotted yellow lines across hepatocytes. Blue rectangle highlights the fluorescence distributed near cell nuclei. l Hepatocytes were incubated with Cy5-mRNA-encapsulated Rhob-PE-labeled LNPs for 1 h in 10% serum. Lyso-endosome system was stained with LysoTracker green. The cytoplasmic distribution of LNPs and mRNA were visualized using confocal microscope. m, n Subcellular fractionation of hEPO mRNA from cytoplasm or membrane containing vehicles. m Time-lapse release of free hEPO mRNA into cytoplasm (n = 8). n At 1.5 h after incubation, the distribution of hEPO mRNA in the cytoplasmic compartment and membrane associated compartment was quantified by rt-PCR (n = 4), Student’s T-test. The subcellular isolation was confirmed by membrane associated marker Lamp-1 staining. All data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.