Investigating the functional contributions of phospholipids in selective organ targeting lipid nanoparticles

Abstract

Modular lipid nanoparticles (LNPs) are a promising platform to deliver mRNA to various tissues and cells. Optimization of LNPs for hepatic and extrahepatic tissues often involves substitution of helper lipids or addition of novel lipids not found in conventional four-component LNPs. Among the lipids that comprise LNPs, the functional contributions of phospholipids (PLs) in selective organ targeting (SORT) LNPs remain poorly understood. In this study, we systematically evaluate the roles of PLs within SORT LNPs. Our results demonstrate that PL enrichment enhances cellular transfection efficiency by increasing membrane fusion and endosomal escape. In vivo, we observe that PL-containing SORT LNPs significantly increase protein expression following intramuscular administration in mice, whereas moderate PL inclusion is optimal for intravenous delivery. Cryo-electron microscopy reveals that PL modulation induces distinct morphological rearrangements in LNP structure, which may influence the selective adsorption of plasma proteins, an essential factor in endogenous targeting mechanisms. These findings highlight the fundamental role of PLs in supporting intracellular delivery and guiding organ-specific biodistribution through protein corona formation. A deeper understanding of the structural and functional impact of lipid components, especially PLs, will be crucial for the rational design of next-generation mRNA delivery systems with improved efficacy and precision.

Keywords: Endogenous targeting; Endosomal escape; Helper lipids; Lipid nanoparticles; Phospholipids; Selective organ targeting (SORT); mRNA delivery.

Fig. 1.. Design and characterization of phospholipid (PL) titration series in organ-selective SORT LNPs.

(a) Schematic of the formulation process for liver-, lung-, and spleen-targeting SORT LNPs. A lipid ethanol mixture (including ionizable lipid, cholesterol, phospholipid, PEG lipid, and SORT lipid) was rapidly mixed with mRNA in an acidic aqueous buffer to form LNPs. (b) Visual summary of PL titration strategy for each organ-targeted LNP. Original SORT formulations are highlighted in bold. PL molar content was systematically increased while compensating by reducing SORT lipid content to maintain a consistent 5-component formulation. (c) Hydrodynamic diameter and polydispersity index (PDI) measured by dynamic light scattering (DLS) for all PL titration series (n = 3). (d) mRNA encapsulation efficiency of PL-titrated SORT LNPs, measured via RiboGreen assay (n = 4).

Fig. 2.. PL incorporation enhances endosomal membrane fusion in a formulation-dependent manner.

(a) Schematic representation of the membrane fusion assay using FRET-labeled plasma membrane-mimicking liposomes (PMML) and endosomal membrane-mimicking liposomes (EMML). Membrane fusion efficiency of liver (b), lung (c), and spleen (d) SORT formulation series, measured by FRET, showing differences in fusion efficiency when tested against PMML and EMML. Data were analyzed via simple linear regression at 120 min post-incubation (n = 4). Results are shown as mean ± SD.

Fig. 3.. Phospholipid inclusion in SORT LNPs boosts in vitro cell transfection efficiency.

(a) Experimental schematic of the in vitro transfection procedure, with measurement of transfection efficiency and cell viability. Quantification of mean luminescence following transfection with liver (b), lung (c), and spleen (d) SORT LNPs in HEK293 (top) and HeLa (bottom) cells (n = 4). Statistical significance was determined using one-way ANOVA with multiple comparisons. The original SORT LNP formulation (bolded) is compared to all other formulations. ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05.

Fig. 4.. Phospholipid enrichment in SORT LNPs improves endosomal escape in cells.

(a) Experimental schematic for in-cell tracking of LNP endosomal escape, measured by the evolution of fluorescence intensity over time using live-cell imaging. Representative data showing endosomal escape for liver (b), lung (c), and spleen (d) SORT LNP series. Left panels show microscopy images (scale bar = 100 μm) of DiD fluorescence intensity at 6 h post-treatment (20 × magnification). Middle panels present the quantified mean fluorescence intensity normalized to cell count per frame (n = 4) as a function of time, with statistical significance indicated at the 6-h timepoint. Right panels show area under the curve analysis of fluorescence intensity over time. Statistical significance was assessed using one-way ANOVA with multiple comparisons, comparing all formulations to the original SORT LNP (bolded). ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05.

Fig. 5.. Phospholipid enrichment in SORT LNPs enhances protein production in vivo.

(a) Experimental schematic depicting in vivo transfection in C57BL/6 mice. Mice were administered SORT LNPs containing hEPO mRNA (dose of 0.3 mg/kg) via intravenous (I.V.) or intramuscular (I.M.) injection. Blood samples were collected at 0, 6, 24, 48, and 72 h post-injection to quantify hEPO levels using ELISA. (b–d) Protein expression kinetics following I.V. (left) and I.M. (right) administration of liver (b), lung (c), and spleen (d) SORT LNP series. Graphs show hEPO levels over time, with area under the curve (AUC) analysis used to determine total protein expression. Statistical comparisons were performed using one-way ANOVA with multiple comparisons, comparing all formulations to the original SORT LNP (bolded). ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05.

Fig. 6.. Phospholipid content affects SORT LNP morphology and protein corona composition.

Cryo-electron microscopy images of Luc mRNA-loaded liver, lung, and spleen SORT LNPs (a). Cryo-electron microscopy images of empty liver (b), lung (c), and spleen (d) SORT LNPs across varying phospholipid compositions, illustrating internal and external structural changes driven by PL enrichment (scale bar = 50 nm). (e) Schematic of the experimental workflow for protein corona analysis via mass spectrometry following incubation of SORT LNPs in purified mouse plasma. Quantification of key adsorbed plasma proteins (ApoE, Vitronectin, and β-2-glycoprotein 1) on liver (f), lung (g), and spleen (h) SORT LNPs. Statistical analysis was performed using one-way ANOVA with multiple comparisons, comparing each formulation to the original SORT LNP (bolded). ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05.