Liposomal lipid nanoparticles for extrahepatic delivery of mRNA

Abstract

Long-circulating, transfection-competent lipid nanoparticle (LNP)-mRNA delivery systems are critical for achieving efficient transfection in extrahepatic tissues. Here we investigate the properties of LNP mRNA systems containing high proportions of bilayer forming lipids, using equimolar egg sphingomyelin and cholesterol as the bilayer-forming components. We show that LNP mRNA systems prepared at bilayer lipid to ionizable lipid molar ratios of 4-0.67 exhibit high mRNA encapsulation efficiencies (90-100%) and excellent transfection potencies in vitro. Systems with bilayer lipid to ionizable lipid molar ratios equating to 4 exhibit a liposomal morphology with a solid core suspended in an aqueous interior surrounded by a lipid bilayer. These liposomal LNPs exhibit longer circulation lifetimes than LNP systems with Onpattro-like lipid compositions and have enhanced extrahepatic transfection properties. The prolonged blood circulation lifetime is attributed to reduced plasma protein adsorption. The transfection competency of liposomal LNP systems is attributed to export of the solid core containing mRNA from the LNP as the endosomal pH is lowered.

Fig. 1. Morphological and in vitro transfection properties of LNP mRNA systems formulated using various bilayer lipid to ionizable lipid molar ratios (R_B/I).

LNP mRNA systems were formulated as described in Methods containing NanoLuc mRNA (808 nt; N/P = 6) with lipid compositions nor-MC3/ ESM: cholesterol/ PEG-DMG at varying ratios of bilayer lipid (ESM and equimolar cholesterol) to ionizable lipid (R_B/I). These formulations correspond to R_B/I of 9, 4, 2.3, 1.5, 1, 0.67 and 0.43. The Onpattro-like formulation consists of MC3/DSPC/cholesterol/PEG-DMG (50/10/38.5/1.5 mol/mol). If it is assumed that the cholesterol and DSPC reside exclusively in the monolayer or bilayer environments, the R_B/I for this formulation is 1.03. A Lipids in ethanol are mixed with mRNA in 25 mM NaOAc (pH 4) to give rise to LNP mRNA systems that are either liposomal LNP systems or solid core systems where at least part of the exterior surface consists of a lipid monolayer. B Encapsulation efficiencies of LNP NanoLuc mRNA systems with varying R_B/I values (mean ± S.D., n = 3). C Hydrodynamic diameter (DLS measurement, number mean) for various R_B/I ratios (mean ± S.D., n = 3). D The classification of LNP morphologies within the formulated population. LNP morphology was defined by its oil core or lamellar/multilamellar liposomal features as determined from cryo-TEM micrographs. E Cryo-TEM micrographs of LNP mRNA systems prepared using various R_B/I values, micrograph has been reproduced twice. F Luminescence of Huh7 cells incubated for 24 h with LNP NanoLuc mRNA systems (0.1–1 μg mRNA/mL), decrease in R_B/I indicated from beige to dark red, with Onpattro-like formulation (indicated as gray) over a range of R_B/I values (mean ± S.E.M., n = 6). Source data are provided as a Source Data file. Figure 1A was partially created in BioRender. Cheng, M. (2025) https://BioRender.com/o07c413. All LNP illustrations are original and were created using Inkscape.

Fig. 2. Liposomal LNP NanoLuc mRNA systems (R_B/I = 4) exhibit enhanced lymphatic and pancreatic gene expression in vivo as compared to LNP with the Onpattro-like lipid composition.

Liposomal LNP (nor-MC3/ ESM/cholesterol/ PEG-DMG; 20/40/40/1.5 mol/mol; R_B/I = 4) and LNP with the Onpattro-like composition (MC3/DSPC/cholesterol/DMG-PEG: 50/10/38.5/1.5 mol/mol) containing NanoLuc (N/P = 6) were prepared and administered i.v. to CD-1 female mice. A IVIS bioluminescence images expressed as radiance (photons/second/cm²/steradian) at various times following injection of LNP mRNA systems (0.5 mg mRNA/kg). B Ex vivo bioluminescence (24 hpi) in heart, lung, liver, spleen, kidney, skin, fat, muscle (left flank), inguinal lymph node and pancreas. C Ex vivo transfection profile of homogenized tissue measured in relative luminescence units/gram of tissue (Grey: Liver; Pink: Spleen; Green: Lymph node; Blue: Pancreas). [More detailed statistical analyses can be found in Fig. S4]. D Representative 3D rendered SPECT/CT reconstructed images of CD-1 mice injected i.v. with either the ¹¹¹In-labelled Onpattro-like LNP formulation or the liposomal LNP NanoLuc mRNA system (∼0.4-0.5 mCi/mouse [¹¹¹In] and ∼15 µg mRNA/mouse corresponding to 0.5 mg mRNA/kg) as a function of time post-injection. E LNP biodistribution as detected by gamma counting of liposomal LNP containing NanoLuc compared to the Onpattro-like composition ex vivo, measured in relative % injected dose/gram (%ID/g) (Grey: Liver; Red: Spleen; Green: Lymph node; Turquoise: Pancreas) [More detailed statistical analyses can be found in Fig. S9 and Table S3]. Source data are provided as a Source Data file.

Fig. 3. Liposomal LNP NanoLuc mRNA system exhibits a similar protein BMC profile but reduced total adsorbed protein compared to LNP with Onpattro-like lipid compositions.

Liposomal LNP (nor-MC3/ ESM: cholesterol/ PEG-DMG; 20/40:40/1.5 mol/mol; R_B/I = 4) and LNP with the Onpattro-like composition (MC3/DSPC/cholesterol/DMG-PEG: 50/10/38.5/1.5 mol/mol), N/P ratio of 6, were formulated, incubated with plasma and isolated as indicated in Methods. A Protein to lipid ratio (wt/wt, µg/µg), total protein measured from a BCA assay, total lipid calculated from DiD-C18 absorbance measurement (mean ± S.D., n = 3). Data were analyzed through a two-tailed unpaired t-test, *p = 0.0296. as indicated in Methods. B SDS–PAGE of the plasma proteins adsorbed to the surface of Onpattro-like and liposomal LNP at 1 hour post incubation (mean ± S.D., n = 3). The plasma protein profile is also shown. C Heatmap analysis of the > 1000 proteins identified (iBAQ intensity log) (White-orange indicates the highest abundance while purple-black indicates lower abundance). D The proportions of proteins with distinct biological functions in the protein coronas of LNP mRNA systems with the Onpattro-like lipid composition as compared to the liposomal LNP (Grey: Others; Yellow: Immuno Proteins; Blue: Apolipoprotein; Green: Complement Proteins; Pink: Albumin; Purple: Coagulation Factors). Source data are provided as a Source Data file.

Fig. 4. Studies on the formation and intracellular delivery mechanisms of liposomal LNP mRNA systems.

Liposomal LNP with R_B/I = 4 (nor-MC3/ ESM: cholesterol/ PEG-DMG; 20/40:40/1.5 mol/mol) or R_B/I = 2.3 (nor-MC3/ESM/cholesterol/PEG-lipid; 30/35/35/1.5 mol/mol) were formulated containing Firefly Luciferase mRNA (N/P = 6), n = 3. A Cryo-TEM micrographs generated following the mixing stage at pH 4 (after dialysis against 25 mM NaOAc pH 4 to remove ethanol) and after dialysis against PBS to raise the pH to 7.4, micrograph has been reproduced twice. The white arrows in the pH 4 preparations indicate the presence of electron dense structures interpreted as complexes of mRNA with ionizable lipid. The white arrows in the pH 7.4 micrograph indicate the electron dense oil droplet structures formed by the ionizable lipid in the neutral form. B Cryo-TEM micrographs of LNP mRNA systems (originally in PBS pH 7.4 buffer) that were subsequently dialyzed against lower pH buffers to mimic endosomal pH environments (pH 6.4: early endosomes, pH 5.6: late endosomes, pH 5.0: lysosomes), micrograph has been reproduced twice. The white arrows in the pH 5.6 and pH 5 micrographs show that the electron dense regions decrease in size quickly as the pH is lowered, consistent with a partitioning of the charged form into the lipid bilayer.

Fig. 5. Model for the formation and transfection of liposomal LNP-mRNA systems.

A On mixing an ethanol solution of a lipid mixture containing a high proportion of bilayer-forming lipids and an ionizable lipid with an aqueous buffer (pH 4) containing mRNA, an mRNA cationic lipid complex forms and acts as a nucleation point for subsequent deposition of bilayer lipid that includes residual positively charged ionizable lipid. As the pH is raised to 7.4, the complex moves to the interior of the bilayer protrusion and enlarges due to partitioning of the neutral form of the ionizable lipid into an oil droplet. The mRNA likely dissociates from the oil core at pH 7.4 and resides in the aqueous interior. B Proposed mechanism of transfection of liposomal LNP mRNA systems. Following uptake into an endosome and as the pH of the environment decreases, the ionizable lipid in the oil core moves to the outer monolayer of the LNP bilayer resulting in extrusion of an electron dense region and a positive surface charge on the exterior of the LNP. This encourages close association with the negatively charged endosomal membrane, fusion and release of the mRNA into the cytoplasm. Figure 5 is completely original and was illustrated using Inkscape. All LNP illustrations are original and were created using Inkscape.