Formulation-Driven Optimization of PEG-Lipid Content in Lipid Nanoparticles for Enhanced mRNA Delivery In Vitro and In Vivo

All reagents used in this study were of analytical grade unless otherwise stated. Tris(2-aminoethyl) amine, tetradecyl acrylate, D-fluorescein potassium salt, 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) were obtained from Aladdin (Shanghai, China). Cholesterol was purchased from Sigma-Aldrich (St. Louis, MO, USA). Wheat germ agglutinin Alexa Fluor 488 conjugate (WGA-AF488) and anti-fade mounting medium containing DAPI were purchased from Thermo Fisher Scientific (Waltham, MA, USA). The mRNAs encoding firefly luciferase (mLuc), green fluorescent protein (mEGFP), Cy5-labeled mEGFP, and Cell Counting Kit-8 (CCK-8) were purchased from APExBIO (Huston, TX, USA).

The ionizable lipid was synthesized via a Michael addition reaction between tris(2-aminoethyl)amine and tetradecyl acrylate according to previous reports [23,24]. The synthetic route is illustrated in Figure 1a. Briefly, tris(2-aminoethyl)amine (87.74 mg, ~0.6 mmol) and tetradecyl acrylate (804 mg, ~3 mmol) were added to a 5 mL reaction vial. The mixture was stirred magnetically at 90 °C for 72 h under light-protected conditions. Upon completion, the crude product was purified via column chromatography to yield the desired compound, which was stored at −20 °C in the dark for further use. A small amount of the product was dissolved in deuterated chloroform (CDCl₃) and characterized by H¹-NMR spectroscopy (Figure S1).

Lipid nanoparticles (LNPs) were synthesized using a nanoprecipitation method [25,26]. In brief, ionizable lipid, cholesterol, DMG-PEG, and DOPE were each dissolved in anhydrous ethanol at final concentrations of 100, 25, 50, and 25 mg/mL, respectively. These components were then mixed at a molar ratio of ionizable lipid/cholesterol/DMG-PEG/DOPE = 40:(50−X):X:10, where X ranged from 0.1 to 10. Under continuous vortexing, the organic phase was added dropwise into acetate buffer (200 mM, pH 5.4) to facilitate the self-assembly of LNPs. The resulting nanoparticles were dialyzed and stored at 4 °C for further use. Based on our previous optimization studies [23,25], LNPs and mRNA were mixed at an N/P ratio of 12.5:1 for efficient encapsulation.

The average hydrodynamic diameter, polydispersity index (PDI), and zeta potential of the LNP/mRNA complexes were measured by dynamic light scattering (DLS) using a ZetaSizer NanoZS (Malvern Instruments, Worcestershire, UK). Transmission electron microscopy (TEM) was used to observe the morphology of LNP/mRNA particles after negative staining with 1% uranyl acetate.

The encapsulation efficiency of mRNA within the LNPs was quantified using the Quant-iT™ RiboGreen RNA Assay Kit (Thermo Fisher Scientific). Briefly, RiboGreen stock solution was diluted 1:200 with 1× TE buffer (pH 7.5) and 50 μL was added per well in a black 96-well plate. LNP/mRNA complexes containing 0.1 μg of mRNA were diluted with TE buffer to a final concentration of 0.4 μg/mL, and 50 μL of this dilution was added to the wells containing RiboGreen. To calculate the encapsulation efficiency, free mRNA not formulated with LNPs and at the same concentration was included as total mRNA content, and TE buffer alone served as the negative control. After incubation at room temperature for 10 min, fluorescence was measured using a multimode microplate reader (excitation: 480 nm; emission: 520 nm).

HeLa cells and DC 2.4 cells were cultured at 37 °C in a humidified incubator with 5% CO₂. HeLa cells were maintained in DMEM complete medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin (P/S), while DC 2.4 cells were cultured in RPMI-1640 complete medium with the same supplements.

The cytotoxicity of LNP/mRNA complexes was evaluated in HeLa cells using the Cell Counting Kit-8 (CCK-8) assay. Briefly, HeLa cells were seeded in 96-well plates at a density of 10,000 cells per well and incubated for 24 h. The culture medium was then replaced with fresh medium containing LNP/mRNA complexes at varying mRNA concentrations. After 24 h of incubation, the medium was removed, and CCK-8 reagent diluted 1:10 (v/v) in fresh medium was added to each well. After 30 min of incubation, absorbance at 450 nm was measured using a multifunctional microplate reader. Untreated cells served as the control group, and wells containing only CCK-8 working solution were used as the blank group.

The cellular uptake of LNP/mRNA complexes was analyzed using both flow cytometry and confocal laser scanning microscopy (CLSM). For flow cytometry, HeLa cells were seeded in 24-well plates (50,000 cells/well) and incubated overnight. The medium was replaced with fresh medium containing LNPs loaded with Cy5-labeled mRNA (final RNA concentration: 0.4 μg/mL). After 6 h of incubation, intracellular Cy5 fluorescence intensity was quantified by flow cytometry. For confocal imaging, transfected cells were washed three times with 1× PBS, fixed with 4% paraformaldehyde (PFA) for 15 min, and washed again with PBS. Membranes were stained with WGA-AF488 (2 μg/mL) for 10 min, followed by three PBS washes. Nuclei were stained using DAPI-containing anti-fade mounting medium. Cellular internalization was visualized using confocal laser scanning microscopy.

To assess mRNA transfection efficiency, HeLa or DC 2.4 cells were seeded in 24-well plates at a density of 50,000 cells per well and incubated overnight. The medium was then replaced with fresh medium containing LNP/mEGFP complexes (final mEGFP concentration: 0.4 μg/mL). Cells without LNP/mEGFP treatment served as the blank control group. After 24 h of incubation, green fluorescent protein (EGFP) expression was observed using a fluorescence microscope, and transfection efficiency was quantified by flow cytometry.

All animal experiments were performed in accordance with the Health Guide for the Care and Use of Laboratory Animals of National Institutes. Female BALB/c mice (6–8 weeks old) were purchased from Jinan Pengyue Experimental Animal Co., Ltd. (Jinan, China).

To evaluate the in vivo mRNA delivery efficiency of the LNPs, Luc mRNA LNPs were administered to BALB/c mice via tail vein injection at an mRNA dose of 0.2 mg/kg. After 6 h, D-luciferin was injected subcutaneously at a dose of 200 mg/kg. Ten minutes post-injection, the mice were euthanized, and major organs including the heart, liver, spleen, lungs, and kidneys were harvested. Bioluminescence imaging was then performed using a small animal in vivo imaging system, and luciferase expression was quantified by measuring the luminescent signal intensity in each organ.

To evaluate the biosafety of the LNPs in vivo, BALB/c mice were intravenously injected with LNP/mRNA complexes at an mRNA dose of 0.2 mg/kg. After 24 h, the mice were euthanized, and major organs, including the heart, liver, spleen, lungs, and kidneys, were harvested and fixed in 4% paraformaldehyde (PFA). The fixed tissues were subsequently embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E) for histological analysis. Tissue sections were examined under a light microscope. Untreated mice served as the control group.

All experimental results are presented as the mean ± standard deviation (SD) from at least three independent experiments. The unpaired student’s t-test was used to assess statistically significant differences between groups (* p < 0.05, ** p < 0.01, and *** p < 0.001).

The ionizable lipid was synthesized via a Michael addition reaction (Figure 1a). Lipid nanoparticles (LNPs) containing varying molar ratios of DMG-PEG were subsequently formulated using a nanoprecipitation method (Figure 1b). Due to the strong hydration effect of PEG chains, an excessive amount of DMG-PEG may shield the positive charge of the ionizable lipid, thereby weakening the electrostatic interaction with mRNA and potentially impairing the mRNA-loading capacity of the nanoparticles. To evaluate this effect, we first characterized the mRNA encapsulation efficiency of LNPs with varying DMG-PEG contents. The results showed that all LNP formulations exhibited encapsulation efficiencies greater than 80%, indicating efficient mRNA loading across all tested groups (Figure 2a). These findings suggest that the mRNA-loading capacity was not significantly affected by DMG-PEG content within the tested range, up to 10%.

Next, we specifically evaluated the impact of varying DMG-PEG content on particle size and zeta potential. It has been pointed out that the particle size of LNP depends largely on the components of LNP [27], the preparation method [26,28], and the affinity between the PEGylated lipids and the lipophilic core of LNP [22]. Dynamic light scattering (DLS) was employed to measure both particle size and polydispersity index (PDI). As shown in Figure 2b,c, the DMG-PEG content had only a minor effect on particle size. All LNP formulations exhibited a narrow size distribution, with particle diameters ranging from 180 to 230 nm. Notably, LNPs containing 5% and 10% DMG-PEG showed slightly increased diameters (>200 nm), whereas those with 0.1–1.5% DMG-PEG maintained sizes below 200 nm. Despite these slight differences, the overall impact of DMG-PEG content on particle size remained limited. Transmission electron microscopy (TEM) images further revealed that all of the LNP/mRNA exhibited a uniform spherical morphology (Figures 2e and S2), indicating successful nanoparticle self-assembly. Moreover, the zeta potential measurements revealed that all LNP formulations carried a positive surface charge (Figure 2d), which may facilitate electrostatic interactions with the negatively charged cell membrane, thereby enhancing cellular uptake. With increasing DMG-PEG content, the zeta potential exhibited a decreasing trend, likely due to enhanced surface shielding by the PEG chains.

We further evaluated the in vitro biocompatibility of the LNP formulations using HeLa cells. As shown in Figure 2f, all LNPs exhibited high cell viability, confirming their good biocompatibility. However, a slight decrease in cell viability was observed with increasing DMG-PEG content, suggesting that higher molar ratios of DMG-PEG may pose a potential cytotoxicity risk. These findings highlight the importance of optimizing PEG-lipid content to balance formulation stability and biocompatibility.

The in vitro transfection efficiency of LNP/mRNA formulations with varying DMG-PEG content was evaluated in both HeLa cells and DC 2.4 dendritic cells (Figure 3). As shown in Figures 3a–c and S3a, transfection efficiency in HeLa cells was significantly influenced by the DMG-PEG molar ratio in the LNP formulation. Specifically, transfection efficiency exhibited a biphasic trend: it initially increased with higher DMG-PEG content, peaking at 1.5%, and subsequently decreased as the PEG content continued to rise. At 1.5% and 5% DMG-PEG, the fluorescence intensity of mEGFP expression in HeLa cells was 3.1-fold and 2.3-fold higher, respectively, compared to the LNPs containing 10% DMG-PEG. A similar trend was observed in DC 2.4 cells (Figures 3d–f and S3b), where transfection efficiency was also maximized at a DMG-PEG content of 1.5%. Relative to the 10% DMG-PEG group, fluorescence intensity increased 2.3-fold and 1.67-fold in the 1.5% and 5% DMG-PEG groups, respectively. These results suggest that a moderate amount of DMG-PEG (approximately 1.5%) optimizes LNP-mediated mRNA delivery across different cell types.

To further substantiate our conclusion that 1.5 mol% DMG-PEG offers optimal in vitro transfection efficiency, we performed a kinetic study in which HeLa cells were transfected with LNPs containing different DMG-PEG contents. Luciferase expression was measured at multiple time points (3, 6, 12, and 24 h) using a luciferase assay. As shown in, luciferase expression progressively increased over time for most formulations. Notably, the LNPs with 1.5 mol% DMG-PEG consistently yielded the highest luciferase levels at all time points. These findings provide compelling kinetic evidence that supports 1.5 mol% DMG-PEG as the optimal formulation for in vitro mRNA delivery. Moreover, this kinetic analysis offers valuable complementary data and strengthens the effect of PEG-lipid content on modulating transfection performance over time. Figure S4

To further investigate the underlying mechanism, we assessed the cellular uptake of LNPs loaded with Cy5-labeled mRNA in HeLa cells using flow cytometry and confocal microscopy (Figure 4). The cellular uptake profile from flow cytometry analysis generally mirrored the transfection results, with the highest uptake observed at 1.5% DMG-PEG. Interestingly, although the transfection efficiency decreased at higher DMG-PEG levels (≥5%), the cellular uptake and associated fluorescence intensity remained relatively high, surpassing that of LNPs with lower PEG content (≤0.5%). This phenomenon may be attributed to increased LNP accumulation on the cell surface at higher PEG levels, as indicated by the enhanced peripheral fluorescence signals (highlighted in the white arrows, Figure 4c). Excessive PEGylation is known to hinder intracellular trafficking by impeding cell endocytosis and endosomal escape, ultimately reducing mRNA translation efficiency despite sufficient uptake [18,29]. Confocal fluorescent cell imaging further confirmed that the strongest intracellular fluorescence signal was observed in the 1.5% DMG-PEG group. These findings underscore the importance of fine-tuning PEG content in LNP formulations to balance intracellular uptake and transfection efficiency.

To investigate the effect of DMG-PEG content on in vivo transfection, LNPs encapsulating mLuc mRNA were intravenously administered to mice. As shown in Figure 5, bioluminescence imaging at 6 h post-injection revealed that the fluorescent signals were predominantly localized in the liver and spleen. Among all tested groups, LNPs containing 5% DMG-PEG produced the highest bioluminescent signals in both organs. Specifically, liver signal intensities in the 5% DMG-PEG group were 3.2-fold and 2.7-fold higher than those observed in the 1.5% and 10% DMG-PEG groups, respectively. Similarly, the spleen signals were 3.3- and 5.0-fold higher than those in the 1.5% and 10% groups. For a clearer comparative analysis, taking the 10% DMG-PEG group as a reference, liver fluorescence intensities in the 1.5% and 5% groups decreased by 1.2-fold and increased by 2.6-fold, respectively. In the spleen, fluorescence intensities increased by 1.7-fold and 5.4-fold in the 1.5% and 5% groups, respectively. These results indicate that a fine-tuning of DMG-PEG content, specifically to 5%, markedly enhances LNP-mediated mRNA delivery to key target organs in vivo.

To further support our conclusion that 5 mol% DMG-PEG yields optimal in vivo transfection efficiency, we conducted a kinetic study in which mice were intravenously injected with LNPs containing varying DMG-PEG contents. Luciferase expression was monitored at multiple time points (3, 6, 12, and 24 h) using an IVIS bioimaging system. As shown in, luciferase expression peaked at 6 h post-injection across all formulations. Importantly, the formulation containing 5 mol% DMG-PEG consistently exhibited the highest expression at each time point, reinforcing our conclusion that 5 mol% represents the optimal PEG-lipid content for in vivo mRNA delivery. It is worth noting that, in contrast, the in vitro kinetic profile showed a continuous increase in luciferase expression from 3 to 24 h post-transfection for most LNP formulations. This discrepancy likely arises from fundamental differences between in vitro and in vivo environments, including factors such as cellular uptake dynamics, bioavailability, and degradation pathways. In vivo systems are influenced by additional complexities, such as blood circulation, tissue-specific distribution, immune interactions, and metabolic clearance, all of which can significantly impact the kinetics of mRNA expression. These observations underscore the importance of evaluating LNP performance in both in vitro and in vivo settings to fully understand the impact of PEG-lipid composition on delivery efficiency. Figure S5

Interestingly, the optimal DMG-PEG content for in vivo transfection (5%) was higher than that observed for in vitro transfection (1.5%). This discrepancy may result from physiological barriers in the bloodstream. LNPs with insufficient PEGylation (e.g., 1.5% DMG-PEG) may be rapidly cleared by the mononuclear phagocyte system (MPS), limiting their circulation and bioavailability. Previous studies have shown that upon incubation with 40% mouse serum, approximately 40% and 12% of DMG-PEG molecules remained on LNP surfaces at 2 and 9 h, respectively, indicating that high serum protein concentrations accelerate PEG desorption [22]. Such de-PEGylation can enhance organ accumulation and mRNA delivery efficiency. However, excessively high PEG content (e.g., 10%) may stabilize LNPs too strongly, impeding PEG shedding, cellular uptake, and subsequent transfection, as observed from in vitro cell study. Notably, LNPs with 0.5% DMG-PEG content exhibited preferential accumulation in the spleen, where bioluminescence accounted for over 60% of the total body signal. These findings suggest that simple modulation of the four-component formulation of LNPs can enable partial organ-specific distribution. In our study, enhanced delivery to extrahepatic organs, particularly the spleen, was observed when the PEG-lipid content was reduced to 0.5% or 0.1%. Although the underlying mechanisms remain to be fully elucidated, such tissue-specific distribution may help minimize off-target accumulation and expand the therapeutic potential of mRNA-based treatments.

To further reinforce our conclusion that DMG-PEG content plays a critical role in modulating in vivo LNP performance, we additionally evaluated a standard Pfizer-LNP formulation consisting of the ionizable lipid ALC-0315 (50%) and the phospholipid DSPC (10%). As shown in, the trend observed was consistent with our synthetic LNPs: ALC-0315-based LNPs containing 5% DMG-PEG exhibited the highest mRNA delivery efficiency in mice. It is noteworthy that while 5% DMG-PEG resulted in optimal in vivo transfection in both our synthetic LNPs and the Pfizer-LNP system, the ideal PEG-lipid content may vary depending on the specific ionizable lipids and co-lipids employed in different formulations. Therefore, further studies are warranted to systematically explore and optimize PEG content across diverse LNP compositions. Figure S6

To assess biosafety, a histological examination of major organs was performed after LNP administration (Figure 5c). No obvious pathological abnormalities or tissue damage were observed in any group, indicating that all LNP formulations exhibited good biocompatibility. Furthermore, altering the DMG-PEG content did not induce observable toxicity, supporting the safety of PEG-lipid modulation in vivo.

The data presented in this study are available in this article.