Construction of Spleen-Accumulated Polysorbate 20-Containing Ionizable Lipid Nanoparticles for mRNA Delivery

SM-102 and DMG-PEG2000 were purchased from ShoChem (Shanghai, China) Co., Ltd. Poly-sorbate (PS) 20, 5-(N, N-hexamethylene) amiloride, 2-(p-toluidino)6-naphthalenesulfonic acid (TNS), 1,10dioctadecyl-3,3,30,30-tetramethylindotricarbocyanine iodide (DiR), and dialysis bags (with cutoff molecular weights (WMCO) 3 kDa) were purchased from Sigma-Aldrich (Shanghai, China). Cholesterol and DOPE were purchased from Avanti Polar Lipids (Alabaster, AL, USA). DMEM high glucose with L-glutamine, DNase/RNase-free distilled water and fetal bovine serum (FBS) were purchased from Thermo Fisher (Shanghai, China). FAM/Cy5-labelled mRNA were purchased from GenePharma (Suzhou, China). EGFP mRNA was purchased from APExBIO (Shanghai, China). Hoechst 33342 Staining Solution for Live Cells and Enhanced Cell Counting Kit-8 were purchased from Beyotime Biotechnology (Shanghai, China). Healthy female Balb/c mice (6–8 weeks old) were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing China).

PS20-iLNPs with varying molar ratios were prepared by the vortex mixing method according to the molar ratios detailed in. The lipid ethanol solution, which contained SM-102, cholesterol, DOPE, and PS20, was added to the pH 4 sodium citrate buffer at a volumetric ratio of 1:3 (lipid ethanol solution: citrate buffer) and vortexed rapidly. The resulting mixture was incubated at 37 °C for 1 h, followed by dialysis in sodium citrate buffer (pH 4) for 1 h and subsequently in PBS buffer (pH 7.4) for 3 h. PEG-iLNPs were synthesized using the same protocol but were incubated for 15 min at room temperature and dialyzed in PBS (pH 7.4) for at least 1 h. Table S1

iLNPs@mRNA were prepared via the vortex mixing method likewise. SM-102, cholesterol, DOPE, and PS20 were dissolved in ethanol at a molar ratio of 50%:37.5%:10%:2.5%, while Cy5 or FAM-labeled mRNA was dissolved in 10 mM sodium citrate buffer (pH 4). The solutions were quickly vortexed and mixed at a 1:3 (lipid ethanol solution: RNA citrate buffer) volumetric ratio. The resulting mixture was incubated at 37 °C for 1 h and dialyzed for 4 h.

The hydrodynamic size, polydispersity index (PDI), and zeta potential of the iLNPs were measured by dynamic light scattering (DLS, Zetasizer Nano ZEN5600, Malvern Instruments Limited, Malvern, UK). The mRNA encapsulation efficiency was evaluated using a fluorescence spectrometer. The morphology of PEG-iLNPs@eGFP mRNA and PS20-iLNPs@eGFP mRNA was observed using a transmission electron microscopy (TEM) (JEM-2100, JEOL, Tokyo, Japan) with an acceleration voltage of 200 kV. The iLNPs were deposited on a carbon-coated copper grid and dried under ambient conditions. Negative staining was performed by applying 10 μL of 1% phosphotungstic acid solution to the grid for 60 sec and then air-dried at room temperature.

The apparent pKa values of PS20-iLNPs were determined by 2-(p-toluidino)-6-napthalene sulfonic acid (TNS) fluorescent probe. PS20-iLNPs were prepared and stored in PBS with a concentration of 5 mM total lipids. The 100 μM TNS solution and a series of TNS buffer solutions with pH values from 3 to 10 were prepared. TNS buffer contained 1 μM TNS, 10 mM ammonium acetate, 10 mM morpholineethanesulfonic acid (MES), 10 mM HEPES and 130 mM NaCl. The above solutions were vortex mixed, and the fluorescence intensity was measured by spectrophotometer (Ex 321 nm, Em 445 nm) (Fluorescence spectrometer LS 55, PerkinElmer, Waltham, MA, USA) at room temperature. A sigmoidal best fit analysis was applied to the fluorescence data and the pKa was determined as the pH with half-maximal fluorescence intensity.

Red blood cells (RBCs) were isolated from fresh whole blood collected from C57BL/6 mice via centrifugation at 500× g for 5 min. The isolated RBCs were subsequently washed with phosphate-buffered saline (PBS) buffer (pH 7.4) until the supernatant became clear. The RBCs were resuspended in PBS buffers of pH 7.4 and pH 5.5, respectively. PS20-iLNPs (50 μg) with varying molar ratios of PS20 were added to RBC suspension with an equal number of RBCs. After incubating at 37 °C for 2 h, the RBC solutions were centrifuged at 10,000× g for 5 min. The collected supernatants containing hemoglobin content were added to 96-well plates. The absorbance at 450 nm was measured to evaluate the hemoglobin using the microplate reader. The RBC suspension incubated in PBS buffer (pH 7.4) and Triton-100 (3 wt.%) solution served as negative and positive control, respectively.

A hemolysis assay was performed to evaluate the relationship between endosomal escape efficiency and iLNPs concentration. Different concentrations of iLNPs containing 2.5% PS20 were added to RBC suspension at 37 °C for 2 h. Hemoglobin content was assessed by measuring absorbance at 450 nm using a microplate reader.

The uptake of iLNPs containing different proportions of PS20 was analyzed by flow cytometry. C6 cells were seeded into 6 well cell culture plates with the density of 2 × 10⁵ cells per well and incubated for 24 h (37 °C, 5% CO₂). The cells were incubated with different kinds of PS20-iLNPs@Cy5-mRNA (molar ratio of PS20 is 0.5%, 1%, 1.5%, 2.5%, 5%, respectively) for 4 h, which were detached with trypsin, harvested and resuspended in PBS for the flow cytometry assay and analyzed using FlowJo_V10.

The FAM-labeled mRNA was used to prepare mRNA solutions with concentrations of 6.0 nmol/mL, 4.8 nmol/mL, 3.6 nmol/mL, 2.4 nmol/mL, 1.2 nmol/mL, and 0 nmol/mL. The fluorescence intensity of FAM-mRNA at 490 nm was measured using a fluorescence spectrophotometer to establish a calibration curve correlating fluorescence intensity with concentration. PS20-iLNPs@FAM-mRNA with N/P ratio of 2, 4, 6, 8 and 10 were prepared, and Triton-100 was used to break the membrane at 0 h, 24 h, 48 h and 72 h, respectively. Fluorescence spectrometer was used to detect the fluorescence intensity of each group iLNPs and calculate the encapsulation rate. To verify and analyze the stability, hydrodynamic sizes, PDI and zeta-potentials of iLNPs were measured by DLS.

The PS20-iLNPs@mRNA were prepared as mentioned above. Equal concentrations of eGFP mRNA, PS20-iLNPs@eGFP mRNA, PS20-iLNPs@eGFP mRNA with Triton-100 were, respectively, combined with loading buffer (Solarbio, R1055, Beijing, China) and loaded in 2% agarose gel, whose electrophoresis in 1× TAE for 15 min. The results were detected using Amersham Imager 600 (GE Healthcare, Boston, MA, USA).

The time-dependent stability of PS20-iLNPs and PEG-iLNPs was assessed in the presence or absence of serum. PS20-iLNPs and PEG-iLNPs were added to PBS and PBS with 10% FBS (FBS-PBS), respectively. The iLNPs were diluted with PBS or FBS-PBS at a volume ratio of 1:9 (total lipid 2.5 mg/mL). The iLNPs were incubated at 37 °C for 0.5, 2, 24, 48, and 72 h, then measured by dynamic light scattering (DLS, Zetasizer Nano ZEN5600, Malvern, UK) to monitor the changes in hydrodynamic size, polydispersity index (PDI), and zeta-potential of the iLNPs.

PS20-iLNPs and PEG-iLNPs were added to PBS and PBS with 10% (v/v) bovine serum albumin (BSA-PBS), and then incubated at 37 °C for 72 h. The hydrodynamic size (Hs) of the iLNPs were measured by DLS. The fraction of stealthiness (Fs) was calculated using below equation. Fs value close to 1 indicates that the iLNPs exhibit excellent stealth properties, whereas an Fs value approaching 0 signifies the instability of iLNPs due to the presence of proteins. Fs=(Hs in PBS)(Hs in PBS with BSA)

Cell viability was assessed using the CCK-8 assay. The C6 cells were seeded into 96-well plates at a density of 3 × 10³ cells per well and cultured for 24 h. Each well contained 10 μL of iLNPs and 90 μL of culture medium. PBS treated group was used as the control. After 24 h incubation, 10 μL CCK-8 solution was added to each well and further incubated for a period from 30 to 60 min. The optical density (OD) at 450 nm was measured by microplate reader (ELx-808, BioTek Instruments, Winooski, VT, USA). The cell viability is calculated using below equation. Cell Viability=([(As−Ab)])([(Ac−Ab)])×100% where As is the optical density of cells exposed to iLNPs, Ac is the optical density of PBS group, Ab is the optical density of wells with no cells.

The flow cytometry (BriCyte E6, Mindray, Shenzhen, China) and laser scanning confocal microscopy (LSCM, FV3000, Olympus, Tokyo, Japan) were used to detect intracellular uptake of PS20-iLNPs@Cy5-mRNA. C6 cells were seeded in 6-well plates at a density of 2 × 10⁵ cells per well and incubated for 24 h. After treating with PS20-iLNPs@Cy5-mRNA for 0, 1, 2, 4, 6, 8, and 12 h. The fluorescence signal was examined using flow cytometry (BriCyte E6, Mindray, Shenzhen, China) and analyzed with Flow Jo V10. C6 cells were seeded in cell-culture dishes (Applied Nest, Issaquah, WA, USA) and cultured for 24 h. PS20-iLNPs@Cy5-mRNA was added to the cell-culture dishes and treated for 0, 1, 2, 4, 6, 8, and 12 h. The images were acquired using an Olympus FV 3000 microscope (Tokyo, Japan) after the nuclei were stained with Hochest 33342.

C6 cells were seeded in 6-well plates at a density of 2 × 10⁵ cells per well and incubated for 24 h. After treating with different concentrations of PS20-iLNPs@Cy5-mRNA for 6 h. The uptake of Cy5-labeled mRNA delivered by iLNPs at different concentrations was detected by flow cytometry (Mindray, BriCyte E6) and assessed with Flow Jo V10. C6 cells were seeded in cell-culture dishes and cultured for 24 h. The cells were treated by adding different concentrations of PS20-iLNPs@Cy5-mRNA for 6 h. The nuclei were stained with Hochest 33342 for 15 min, washed three times with PBS, and images were collected using an Olympus FV 3000 microscope.

C6 cells were seeded in 6-well plates at a density of 2 × 10⁵ cells per well and incubated at 37 °C with 5% CO₂ for 24 h, and pretreated with serum-free medium containing inhibitors (sucrose at 450 mM, M-β-CD at 2 mM, and 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) at 10 μg/mL) for 30 min. Subsequently, the cells were incubated with PS20-iLNPs@mRNA for 6 h. After the cells were digested with trypsin, harvested, and resuspended in PBS, the cellular uptake of PS20-iLNPs@Cy5-mRNA under different inhibitor conditions was examined using flow cytometry (Mindray, BriCyte E6) and analyzed with Flow Jo V10.

The transfection efficiency of PEG-iLNPs@eGFP mRNA and PS20-iLNPs@eGFP mRNA was assessed using flow cytometry and laser scanning confocal microscopy (LSCM). C6 cells were seeded in 6-well plates at a density of 2 × 10⁵ cells per well and incubated for 24 h at 37 °C with 5% CO₂. The cells were treated with iLNPs at a mRNA concentration of 30 μg/mL for 48 h. After trypsinization, the cells were harvested, resuspended in PBS, examined by flow cytometry (Mindray, BriCyte E6) and analyzed with Flow Jo V10. C6 cells were seeded in confocal dishes (Nest, Issaquah, WA, USA) and cultured for 24 h. PEG-iLNPs@eGFP mRNA and PS20-iLNPs@eGFP mRNA were added to the cell culture dishes and incubated for 48 h. Images were acquired using an Olympus FV 3000 microscope.

DiR was dissolved in methanol and then mixed into a phospholipid mixture solution dissolved in chloroform. After evaporating under a stream of nitrogen gas, an ethanol solution was added. The resulting mixture was vortexed with sodium citrate buffer solution at a 1:3 (v/v) ratio and incubated. Free DiR was removed by dialysis. DiR-tagged PS20-iLNPs and PEG-iLNPs were tail-vein injected into C57BL/6 mice (0.15 mg lipid per mice), respectively. The mice were sacrificed and heart, liver, spleen, lung, kidney were excised 6 h after the injection, respectively. Eventually, the fluorescence images were acquired by IVIS system (ABL-X5, Tanon, Shanghai, China) to analyze the average fluorescence intensity in these major organs.

All datasets were statistically assessed using GraphPad Prism 10.1.2, with triplicate-independent experiments expressed as mean ± SD. Two-group comparisons employed Student's t-test, whereas multiple comparisons utilized one-way ANOVA. Significance thresholds were defined as: * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

A series of PS20-containing iLNPs (molar ratios ranging from 0.5% to 30%) were prepared via vortex mixing method to optimize the formulation. To ensure endosomal escape of iLNPs, the molar ratios of SM-102 and DOPE were fixed, while cholesterol and PS20 molar ratios were modulated between 39.5 and 10.0% and 0.5–30%, respectively (Table S1). As shown in Figure 1a,b, the size and polydispersity index (PDI) of PS20-iLNPs gradually decreased with increasing PS20 relative molar ratios. At PS20 molar ratios of 1.5–5%, the size of PS20-iLNPs stabilized, while the PDI continued to decrease, implying that the presence of more PS20 enhanced the homogenization of nanoparticles. Conversely, the increase in the relative molar ratio of PS20 from 5% to 30% resulted in larger and heterogeneous nanoparticles (Figure S1). These results indicated that PS20, as an excipient, significantly influenced iLNPs size and homogeneity. The optimal PS20 concentrations range from 1.5% to 5%. iLNPs with varying concentrations of PS20 exhibited a slightly negative zeta potential (~−5 mV) (Figure 1c), enabling the resistance to serum protein adsorption [45], prolonging circulation. pKa value has been demonstrated to determine the surface charge property, which is believed to be related to the facilitated endosomal escape process [46]. The highly efficient intracellular delivery of bioactive macromolecules with optimal values is between 6.0 and 6.5 [47]. Therefore, 2.5% and 5% PS20-iLNPs likely exhibit optimal endosomal escape efficiency (Figure 1d and Figure S2). To validate this hypothesis, the membrane disruption capacity of PS20-iLNPs was quantified via a hemolysis assay. The results show that 2.5% and 5% PS20-iLNPs achieved endosomal escape efficiencies of 81.8% and 69.7%, respectively (Figure 1e). Notably, PS20-iLNPs exhibited negligible membrane disruption ability at physiological pH 7.4 relative to pH 5.5, indicating that iLNPs maintained structural integrity and safety in blood circulation. For RNA payload delivery optimization, formulation screening was conducted through flow cytometric quantification of cellular internalization. As shown in Figure 1f and Figure S3, Cy5-labeled mRNA fluorescence was detected in all groups after being treated with PS20-iLNPs (containing different relative molar ratios of PS20), while the highest fluorescence intensity appeared in the 2.5% PS20-iLNPs group. These findings suggest that 2.5% PS20-iLNPs represent an optimized drug delivery vehicle. For further studies, 2.5% PS20-iLNPs (molar ratio of SM-102, cholesterol, DOPE, and PS20 = 50:37.5:10:2.5) were selected for detailed characterization and mRNA delivery studies.

The stability and stealthiness of the 2.5% PS20-iLNPs (referred to as PS20-iLNPs), were examined using PEG-iLNPs as the control group. After 72 h incubation in PBS with 10% fetal bovine serum (FBS), dynamic light scattering (DLS) results revealed a substantial size increase in PEG-iLNPs group from 117 nm to 215 nm (Figure 2b and Figure S4c,d), exhibiting an 82.6% growth rate, particularly evident at 24h with significant particle enlargement. In contrast, the PS20-iLNPs group exhibited a size increase from 194 nm to 231 nm (Figure 2a and Figure S4a,b), with a growth rate of 19.1%, indicating their superior stability compared to PEG-iLNPs. The anti-protein corona capability of PS20-iLNPs was assessed through adding BSA into the PBS and incubating with iLNPs under physiological conditions (pH 7.0, 37 °C). The hydrodynamic size (Hs) of protein adsorption on both iLNPs were expressed as the fraction of stealthiness (Fs) values. Fs value close to 1 indicated that iLNPs possess excellent stealthiness, whereas Fs value approaching to 0 suggests instability in the presence of proteins. As shown in Figure S5, both formulations demonstrated comparable stealth capacity within the initial 24 h, while from 24 to 72 h, PS20-iLNPs exhibited significantly higher Fs values than PEG-iLNPs (Figure S5 and Figure 2c), highlighting PS20's superior structural stabilization and protein corona mitigation capabilities. PS20-iLNPs exhibited stealth characteristics comparable to those of the zwitterionic polymer poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) as a PEG substitute [37], representing PS20 had the potential to alternative polyethylene glycol (PEG) in biomedical applications.

Safety is a crucial consideration in drug delivery carrier development, as it is directly related to the clinical application potential and patient treatment safety. iLNPs were co-incubated with cells for 24 h, and the safety of PEG-iLNPs and PS20-iLNPs was examined by CCK-8 assay. As shown in Figure 2d,e, both iLNPs showed no toxicity in C6 cells at lower lipid concentrations (0–0.3 mg/mL). At high lipid concentration (0.7–2.7 mg/mL), the cell viability of PS20-iLNPs group exhibited a negligible decrease. Even at the highest concentration (2.7 mg/mL), the cell viability remained at 91.1%, with no observable morphological changes. In contrast, the cell viability of the PEG-iLNPs group was significantly decreased. These findings demonstrate PS20-iLNPs' enhanced biocompatibility relative to PEG-iLNPs across lipid dosage gradients, suggesting dose-dependent therapeutic efficacy optimization through controlled PS20-iLNP administration.

Additionally, we employed a hemolysis assay to evaluate the safety of PS20-iLNPs during blood circulation and to simulate their endosomal escape efficiency in cells. As shown in Figure 2f, erythrocyte hemolysis was minimal in the physiological environment (pH 7.4), demonstrating that PS20-iLNPs can effectively maintain the structural integrity and ensure the safety during blood circulation. It is noteworthy that under the acidic environment (pH 5.5), the hemolysis rate gradually increased with the increase in PS20-iLNPs concentration, suggesting efficient endosomal escape of iLNPs in a concentration-dependent manner. These findings support the potential of PS20-iLNPs for efficient cellular uptake and transfection.

Ionizable lipid-mRNA lipid nanoparticles are formed through electrostatic interactions between the ionizable amine groups of lipid-like polyamines and the phosphate groups of mRNA molecules. The N/P ratio, defined as the molar ratio of nitrogen (in the amine head-groups of lipopolyamines) to phosphorus (in the oligonucleotide backbones), is a critical parameter for evaluating nucleic acid delivery efficiency using lipid nanoparticle [48]. This study aimed to determine the optimal N/P ratio for encapsulating Cy5-labeled mRNA in PS20-iLNPs (PS20-iLNPs@Cy5mRNA). As shown in Figure 3a, the highest encapsulation efficiency (EE%) of 70.2% was at N/P ratio of 6. The PS20-iLNPs@Cy5mRNA at varying N/P ratios were stored at 4 °C for 72 h, the encapsulation efficiency of iLNPs decreased in all groups except for N/P ratio of 6 group (Figure 3b and Figure S6), which indicated that the interactions between the phospholipids and the nucleic acids were the most stable at N/P of 6, ensuring the structural stability of the PS20-iLNPs@Cy5mRNA. Agarose gel electrophoresis analysis demonstrated that eGFP mRNA was effectively encapsulated within PS20-iLNPs@eGFP mRNA, with maintained mRNA integrity (Figure 3c). Further physical characterization revealed that PS20-iLNPs@Cy5mRNA had an average diameter of 199.3 nm. Electrostatically driven interaction between negatively charged mRNA and protonated SM-102 resulted in slightly reduced particle size of PS20-iLNPs@Cy5mRNA than that of bare PS20-iLNPs (Figure 3d). The morphological features of PS20-iLNPs@eGFP mRNA and PEG-iLNPs@eGFP mRNA were visualized using transmission electron microscopy (TEM), demonstrating nanoparticles with near-spherical topography and well-defined peripheral boundaries. (Figure 3e,f). Under physiological pH, both loaded and bare PS20-iLNPs exhibited near-neutral zeta-potentials (−2.4467 mV), demonstrating that the mRNA was successfully encapsulated in iLNPs (Figure 3g).

Given the critical role of efficient intracellular uptake and delivery of PS20-iLNPs in genetic therapy, the cellular internalization of PS20-iLNPs@Cy5mRNA and PEG-iLNPs@Cy5mRNA were quantitatively analyzed by flow cytometry. We incubated both iLNPs with the C6 cells for 4 h in the presence of serum. PS20-iLNPs@Cy5mRNA showed a 25.9% increase in mean fluorescence intensity (82,526 vs. 65,545) compared to PEG-iLNPs@Cy5mRNA (Figure 3h,i). This may be attributed to the superior anti-protein adsorption capability of PS20-iLNPs (consistent with Figure 2c), which reduces protein corona formation to enhance cellular uptake.

To elucidate the mRNA delivery mechanism of PS20-iLNPs, we investigated the cellular uptake patterns and pathways of the optimal formulation PS20-iLNP. Using Cy5-labeled mRNA, we observed that PS20-iLNPs@Cy5mRNA was significantly internalized by cells after 4 h incubation (Figure 3l). Furthermore, the uptake efficiency increased with prolonged incubation time (Figure 3j and Figure S7b). Based on these findings, we fixed the interaction time of cells-iLNPs at 6 h. After a 6 h incubation in same amount cells with varying iLNPs concentrations, the minimum concentration for effective cellular uptake of PS20-iLNPs@Cy5mRNA was determined to be 80 μg/mL (Figure 3m). Intracellular fluorescence intensity (FI) increased with higher administration concentrations, confirming concentration-dependent cellular uptake (Figure 3k and Figure S7a). Next, we investigated the endocytic pathways using sucrose, EIPA and M-β-CD, which are inhibitors to clathrin-mediated endocytosis, macropinocytosis and caveolae-mediated endocytosis, respectively. For PEG-iLNPs, M-β-CD and sucrose significantly reduced cellular uptake by 70.4% and 61.5%, respectively (Figure S8a,b), suggesting that caveolae-mediated and clathrin-mediated endocytosis are the primary pathways. However, in PS20-iLNPs group, EIPA and sucrose inhibited the cellular uptake efficiency by 46.2% and 20%, respectively (Figure S8c,d), which indicated that PS20-iLNPs are different from PEG-iLNPs; they are mainly internalized via macropinocytosis, and lesser via clathrin-mediated endocytosis. Despite employing distinct internalization pathways, the two iLNP formulations exhibit comparable intracellular distribution after entry (Figure S9).

The transfection efficiency was evaluated using the expression levels of eGFP mRNA encapsulated in PEG-iLNPs (PEG-iLNPs@eGFP mRNA) and PS20-iLNPs (PS20-iLNPs@eGFP mRNA). After transfecting C6 cells for 48 h, the expression of eGFP mRNA was assessed by confocal microscopy and flow cytometry. As shown in Figure 4a, confocal microscopy revealed that both PS20 and PEG group can effectively deliver eGFP mRNA into cells, preserving mRNA biological function and enabling successful expression. Figure S10 indicated that the transfection ability of PS20-iLNPs was higher than PEG-iLNPs by analyzing the fluorescence signals of Figure 4a using ImageJ. Subsequently, the delivery efficiency of both iLNPs was further quantified by flow cytometry. Figure 4b showed that the eGFP expression in PS20 group was slightly higher than PEG group. Quantitative analysis showed that the mean eGFP fluorescence intensities of PEG and PS20 group were 43,388 and 52,075, respectively, which were consistent with the trend of Figure S10. The transfection efficiency of PS20 group was 46.5%, which was 10.6% higher than that of PEG group (Figure 4c). Polysarcosine (pSar) has been demonstrated to serve as a polyethylene glycol (PEG) alternative for lipid nanoparticle (LNP) formulation [40]. However, the mRNA expression with the pSar systems was lower than with the PEG-based lipid nanoparticle [40]. PS20-iLNPs achieved superior transfection performance than PEG-iLNPs, which indicated the enhanced efficacy of PS20 over pSar as the PEG substitute in iLNPs gene delivery systems.

Collectively, these data establish PS20 as a multifunctional excipient that inhibits protein corona formulation, improves biocompatibility, and facilitates efficient mRNA delivery, leading to functional protein translation. This formulation represents a viable PEG-free strategy for advanced nanoparticle engineering.

Given the significance of systemic mRNA delivery in numerous applications, including protein replacement therapy and systemic immunization, we further investigated the distribution characteristics of PS20-iLNPs drug delivery system in animals following systemic injection. DiR-labeled iLNPs were intravenously injected into C57BL/6 mice via the tail vein, and the mice were sacrificed 6 h post-injection for isolation and examination of major organs. The fluorescence signals of isolated organs were recorded by in vivo imaging system to monitor the distribution of PS20-iLNPs. The result of fluorescence signals indicated that iLNPs were mainly accumulated in the liver and spleen (Figure 5a). Notably, the fluorescence intensity statistical analysis exhibited that PS20-iLNPs possess significantly higher infiltration to spleen than PEG-iLNPs (Figure 5b), with approximately 24-fold higher accumulation. While the gangliosides-based LNPs developed by Permana et al. exhibited stealth properties and protein resistance comparable to PEG-based LNPs, their biodistribution demonstrated dual hepatic/splenic accumulation, which could potentially exacerbate drug-induced hepatotoxicity [42]. In contrast, PS20-iLNPs achieved spleen-specific accumulation without elevating hepatic metabolic burden, maintaining safety with precision delivery. The splenic accumulation mechanism of PS20-iLNPs is primarily attributed to their optimized particle size (199.3 ± 1.5 nm). This dimension aligns with the structural features of splenic venous sinusoids, where the reticuloendothelial cell gaps are approximately 200–500 nm [49]. Numerous studies have demonstrated that nanoparticles within the 200–500 nm range exhibit preferential splenic retention due to mechanical filtration mechanisms [50,51,52,53]. Moreover, Zhang et al. conducted comparative analyses revealing that 215 nm nanoparticles achieved superior splenic enrichment relative to 165 nm and 285 nm counterparts, thereby establishing 200 nm as an optimal threshold for efficient splenic targeting [54]. On the other hand, owing to the constitutive activity of macropinocytosis in immature dendritic cells (DCs), particles of relatively larger sized (e.g., ≥200 nm) are advantageous for DC targeting [55,56,57]. Our PS20-iLNPs size of ~200 nm may explain the spleen accumulate property (Figure 5).