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International Journal of Pharmaceutics: X logoLink to International Journal of Pharmaceutics: X
. 2026 Apr 29;11:100553. doi: 10.1016/j.ijpx.2026.100553

Engineering a safe and potent LNP-mRNA delivery system by leveraging the dual activities of α-tocopherol

Huijun Wang a,d,1, Ziyao Kang a,e,1, Xiaoshuang Wang b,e,1, Zheng Pan a,d, Yun Sun a,d, Yuan Li a,d, Xin Li b,d, Shuai Shao c,d, Xiang Zheng c,d, Zibo Han b,d,⁎, Jin Ren a,d,⁎
PMCID: PMC13158621  PMID: 42125467

Abstract

The widespread application of lipid nanoparticles (LNPs) as mRNA delivery vectors is constrained by the intrinsic trade-off between delivery efficacy and inflammatory reactogenicity. To address this limitation, we engineered a safe and potent LNP-mRNA delivery system by leveraging the dual activities of α-tocopherol (TP). Capitalizing on its well-documented anti-inflammatory and immunomodulatory properties, we hypothesized that incorporating TP or its derivatives into LNPs would concurrently mitigate carrier-induced inflammation and enhance antigen-specific immunogenicity. Our results demonstrated that TP succinate (TPS)-modified LNPs significantly improved in vitro mRNA delivery, achieving up to a 9.5-fold increase in protein expression alongside enhanced cellular uptake, without compromising biocompatibility. Following immunization, TPS-LNPs markedly reduced acute inflammatory reactogenicity, as evidenced by a threefold lower serum IL-6 level at 6 h compared to the standard formulation in blank-LNP groups, and by significantly attenuated cytokine levels in mRNA-loaded groups. In vivo, TPS-LNPs elicited robust and balanced immune responses, characterized by potent humoral immunity and enhanced antigen-specific T cell activation. Mechanistically, the enhanced immunogenicity was associated with upregulated CD40 expression on antigen-presenting cells. Importantly, the anti-inflammatory attributes of TP derivatives conferred an excellent safety profile, with no evidence of significant tissue damage or systemic toxicity. Our findings advocate for a functionalization paradigm in LNP design, wherein α-tocopherol derivatives serve as intrinsic modulators to recalibrate innate immunity, thereby proposing a new design benchmark for simultaneously safe and potent mRNA delivery.

Keywords: Lipid nanoparticles, α-Tocopherol, Immunomodulation, Anti-inflammatory, mRNA delivery

Graphical abstract

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1. Introduction

Messenger RNA (mRNA) has emerged as a transformative therapeutic modality, holding immense promise not only in vaccination but also in areas such as protein replacement therapy and genome editing (Barbier et al., 2022; Qin et al., 2022). The clinical success of mRNA is inextricably linked to the development of safe and effective delivery systems (Pardi et al., 2018; Xiong et al., 2018). Among these, lipid nanoparticles (LNPs) have become the leading non-viral platform, underpinning the efficacy of all currently approved mRNA vaccines (Hou et al., 2021; Paunovska et al., 2022; Cullis and Felgner, 2024). Conventional LNPs are typically formulated with four components: an ionizable lipid for mRNA complexation, a helper phospholipid for structural integrity, cholesterol for membrane stability, and a polyethylene glycol (PEG)-lipid to modulate nanoparticle pharmacokinetics (Xu et al., 2025; Eygeris et al., 2022; Tenchov et al., 2023; Albertsen et al., 2022). Despite their foundational role, first-generation LNPs face a persistent and intrinsic challenge: the trade-off between delivery potency and inflammatory reactogenicity (Wadman, 2020; Rosenblum et al., 2022; Wang et al., 2024). The cationic nature of ionizable lipids, crucial for mRNA encapsulation and endosomal escape, can also trigger robust and sometimes excessive innate immune responses. This manifests as local injection site reactions and systemic inflammatory symptoms, which, while often transient, may limit patient tolerance and impede dose escalation for therapeutic applications beyond vaccines (Ndeupen et al., 2021; Sharma et al., 2024). Although attempts to mitigate this reactogenicity through modifications of lipid chemistry and adjustments to component ratios have yielded incremental improvements, the fundamental need to simultaneously achieve high delivery efficiency and low reactogenicity persists, continuing to drive innovation in LNPs design. Emerging strategies, such as the incorporation of functional fifth components, stimuli-responsive lipids, or immune-modulatory moieties, aim to decouple these traditionally opposing properties, thereby expanding the therapeutic window of mRNA-LNPs platforms for chronic and precision medicine applications (Han et al., 2024; Vadovics et al., 2025; Lu et al., 2023; Cheng et al., 2020).

In this context, α-tocopherol (TP), the primary form of vitamin E, presents a uniquely attractive solution due to its dual biological activities. TP is widely recognized as a potent anti-inflammatory agent, capable of scavenging reactive oxygen species and suppressing key pro-inflammatory signaling pathways such as NF-κB (Pan et al., 2025; Wallert et al., 2019). Paradoxically, and crucially for vaccine applications, TP is also a documented immunomodulator (Salinthone et al., 2013; Alimdzhanovna, 2025). It is a key functional component of the licensed adjuvant AS03, where it enhances vaccine immunogenicity by promoting cytokine and chemokine production, facilitating antigen uptake, and recruiting immune cells to draining lymph nodes (Lodaya et al., 2019; Morel et al., 2011; Pulendran et al., 2021). This rare combination of properties suggests that TP is not merely a passive antioxidant but an active biological modifier capable of “calibrating” the immune response—dampening harmful, non-specific inflammation while promoting productive, antigen-specific adaptive immunity. We hypothesized that integrating α-tocopherol or its derivatives directly into the LNP matrix could yield a dual-functional delivery system. We posited that these molecules would act from within the nanoparticle to simultaneously achieve two objectives: (i) leveraging their anti-inflammatory activity to quench LNP-induced oxidative stress and innate immune overactivation, thereby improving systemic and local safety; and (ii) harnessing their immunomodulatory (adjuvant-like) activity to enhance antigen presentation and lymphocyte priming, thereby boosting therapeutic potency. To test this hypothesis, we developed a series of LNPs modified with α-tocopherol (TP), its hydrolyzable succinate ester (TPS), and its water-soluble PEGylated derivative (TPGS). We systematically evaluated their impact on the physicochemical properties of LNPs, mRNA delivery efficiency in vitro, and the immunogenicity and safety profiles in vivo using a model antigen. Here, we demonstrate that integrating α-tocopherol succinate as a structural and functional component within LNPs not only attenuates carrier-induced inflammation but also enhances antigen-specific immunogenicity, a dual functionality that effectively decouples the classical efficacy–safety trade-off. This study repositions α-tocopherol from a mere excipient to a core design element for biomaterials, paving the way for a new class of “self-adjuvanting” and “reactogenicity-tuned” mRNA delivery systems with broad therapeutic applicability.

2. Materials and methods

2.1. Materials

Lipids and chemical reagent: Heptadecan-9-yl8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (SM-102), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and methoxypoly (ethylene glycol) dimyristoyl glycerol (DMG-PEG2000) were purchased from Xiamen Sinopeg Biotech Co., Ltd. (China). Cholesterol was obtained from Sigma-Aldrich (USA). α-Tocopherol (TP) and α-tocopheryl succinate (TPS) were sourced from Yuanye Bio-Technology (China) and Shanghai Macklin Biochemical Co., Ltd. (China), respectively. α-Tocopherol polyethylene glycol succinate (TPGS) was obtained from Shanghai Chineway Pharma Tech Co., Ltd. (China). Amiloride was purchased from Shanghai Acmec Biochemical Technology Co., Ltd. (China). Chlorpromazine hydrochloride was obtained from Beijing Solarbio Science & Technology Co., Ltd. (China). Methyl-β-cyclodextrin (average Mw 1310) was sourced from Acros Organics (Germany). Sucrose was purchased from AVT (Shanghai) Pharmaceutical Tech Co., Ltd. (China).

Biochemical reagents and antibodies: Absolute ethanol was acquired from Sinopharm Chemical Reagent Co., Ltd. (China). Bovine serum albumin (BSA), Tween-20, and 4% paraformaldehyde were purchased from Beyotime Biotech Co., Ltd. (China). Respiratory syncytial virus (RSV) mRNA was custom-synthesized by Levostar Biotech Co., Ltd. (China). Firefly luciferase and enhanced green fluorescent protein mRNA were purchased from Novoprotein Scientific Inc. Fluorescently conjugated antibodies for flow cytometry were purchased from BD Biosciences (USA), BioLegend (USA), and Thermo Fisher Scientific (USA), as detailed in Appendix A.

2.2. Cells and animals

The HEK-293 T cell line was procured from the National Biomedical Cell-Line Resource (NSTI-BMCR, China) and maintained in Dulbecco's Modified Eagle Medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Every Green, China) and 1% penicillin-streptomycin (HyClone) at 37C with 5% CO₂. HepG2 cells, also obtained from the National Biomedical Cell-Line Resource (NSTI-BMCR, China), were cultured in Minimum Essential Medium (MEM, Gibco) supplemented with 10% FBS and 1% penicillin-streptomycin under the same incubation conditions. Female specific-pathogen-free (SPF) BALB/c mice (4–8 weeks old) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (China). All animal procedures were performed in compliance with the Chinese Regulations for the Administration of Laboratory Animals and the ARRIVE guidelines, and were approved by the Animal Experiment Committee of the National Vaccine and Serum Institute (Assurance Number: NVSI-RCD-JSDW-ER-2024309).

2.3. Preparation of dual-functional LNPs

For in vitro studies, conventional and modified LNPs were prepared via the ethanol dilution method (Su et al., 2024). The standard formulation (Std-LNPs) comprised SM-102, DSPC, cholesterol, and DMG-PEG2000 at a molar ratio of 50:10:38.5:1.5. To engineer dual-functional LNPs, α-tocopherol (TP), α-tocopheryl succinate (TPS), or TPGS was incorporated as a fifth component at specified mass ratios, partially replacing the standard lipids (detailed in Fig. 1A-B). In one formulation, DMG-PEG2000 was fully substituted with TPGS. The mRNA was dissolved in 20 mM citrate buffer (pH 4.5), while the lipid components (SM-102, DSPC, cholesterol, DMG-PEG2000, and the respective TP derivatives) were dissolved in absolute ethanol. Subsequently, the ethanol phase was rapidly injected into the aqueous mRNA solution at a final ethanol-to-aqueous phase volume ratio of 1:3. The mixture was vortexed for 30 s and then incubated at 25 °C for 10 min to allow for LNPs formation. The LNP suspension was buffer-exchanged into phosphate-buffered saline (PBS, pH 7.4) using a 30-kDa molecular-weight cut-off (MWCO) centrifugal filter device (Merck Millipore) to remove residual ethanol and non-encapsulated components. For in vivo applications, LNPs were prepared using an Ignite™ microfluidic system (NanoAssemblr™, Cytiva). Purification was achieved by centrifugal filtration (30 kDa MWCO; Merck Millipore) and buffer exchange into PBS (pH 7.4) (Chen et al., 2016).

Fig. 1.

Fig. 1

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Composition and physicochemical characterization of α-tocopherol-modified lipid nanoparticles (LNPs). (A) Pie charts depicting the different formulations for ionizable lipid (SM-102), phospholipid (DSPC), cholesterol, PEG lipid (DMG-PEG2000), and α-tocopherol (TP) and its derivatives. (B) Formulation details of α-tocopherol-modified LNPs. (C) Hydrodynamic diameter, polydispersity index (PDI), and zeta potential of the formulated LNPs. (D) mRNA encapsulation efficiency of different LNPs. Data are presented as mean ± standard error of the mean (SEM).

2.4. Quantification of α-tocopheryl succinate in LNPs by high-performance liquid chromatography

To verify the actual α-tocopheryl succinate (TPS) content in the modified LNPs, TPS was quantified using high-performance liquid chromatography coupled with charged aerosol detection (HPLC-CAD) method (Yu et al., 2023). Briefly, 10% and 20% TPS-modified LNPs were lysed with a lysis solution consisting of ethanol and formamide (85:15, v/v) to release the lipid components. Chromatographic separation was performed on an Acclaim™ 300 C18 column (3 μm, 4.6 × 150 mm, Thermo Fisher Scientific) maintained at 50 °C. The mobile phase consisted of two eluents delivered in gradient mode at a flow rate of 0.8 mL/min, and the injection volume was 10 μL. The charged aerosol detector (CAD) was set with an evaporation temperature of 50 °C, a data acquisition frequency of 10 Hz, and a filter constant of 3.6 s. TPS concentration was determined from a standard calibration curve (2–80 μg/mL, R2 > 0.99). The actual TPS content was calculated as the molar ratio of TPS to total lipids.

2.5. Physicochemical characterization of LNPs

The hydrodynamic diameter, polydispersity index (PDI), and zeta potential of LNPs were measured by dynamic light scattering (DLS) and electrophoretic light scattering using a Zetasizer Nano ZS90 (Malvern Panalytical, UK). The encapsulation efficiency (EE) of mRNA was determined using the Quant-iT RiboGreen RNA Assay Kit (Invitrogen, USA). Samples were divided into two aliquots: one was treated with 1× TE buffer to measure free mRNA, and the other was lysed with 2% Triton X-100 in TE buffer to release total mRNA. After adding the RiboGreen reagent, fluorescence was measured using a SpectraMax M5 microplate reader (Molecular Devices, USA) at excitation/emission wavelengths of 480/520 nm. mRNA concentrations were calculated from an RNA standard curve. EE was calculated as (encapsulated mRNA/total mRNA) × 100%.

2.6. Assessment of transfection efficiency of LNPs in vitro

The delivery efficiency of LNPs was evaluated in HEK-293 T cells using mRNA encoding either firefly luciferase or enhanced green fluorescent protein (eGFP). For luciferase expression, cells were seeded in white-bottom 48-well plates at a density of 4.0 × 104 cells per well. After 24 h, cells were transfected with LNPs containing 0.5 μg of luciferase mRNA. Luciferase activity was quantified 24 h post-transfection using a commercial assay kit (Yeasen, China) according to the manufacturer's instructions. For eGFP expression analysis, cells were seeded in 24-well plates at 1.0 × 105 cells per well and transfected with LNPs containing 1.5 μg of eGFP mRNA. After 24 h, cells were harvested, and the mean fluorescence intensity (MFI) was analyzed by flow cytometry (FACSCanto II, BD Biosciences, USA).

2.7. Evaluation of cellular uptake of LNPs in vitro

Confocal microscopy analysis of cellular uptake: LNPs were labeled with the lipophilic fluorescent dye DiD (Vybrant DiD, Invitrogen, USA) by incubating the dye with the LNP formulation at room temperature for 15 min. Unbound dye was removed by ultrafiltration (30 kDa, Merck Millipore). HEK-293 T cells were seeded in 35 mm confocal dishes and incubated with DiD-labeled LNPs for 4 h. Cells were then fixed with 4% paraformaldehyde (Servicebio, China), and nuclei were counterstained with DAPI (Beyotime, China). Cellular internalization of LNPs was visualized using a confocal laser scanning microscope (LSM 980, Carl Zeiss, Germany).

Flow cytometric analysis of cellular uptake: To investigate the endocytic pathways involved in LNPs internalization, HEK-293 T and HepG2 cells were seeded in 12-well plates at a density of 1 × 105 cells per well and incubated overnight. Cells were pre-incubated with various endocytosis inhibitors for 30 min at 37 °C prior to the addition of DiD-labeled LNPs. After 2 h of co-incubation, cells were washed three times with PBS, detached with trypsin-EDTA, and resuspended in PBS. Fluorescence intensity was measured using a flow cytometer (FACSCanto II, BD Biosciences, USA), and data were analyzed using FlowJo software (BD Biosciences, USA).

2.8. In vitro cytotoxicity assay of LNPs

The cytotoxicity of LNPs was assessed using the Cell Counting Kit-8 (CCK-8, Dojindo, Japan). HEK-293 T cells were seeded in 96-well plates and treated with LNPs encapsulating eGFP mRNA (0.25 μg per well) for 24 h. Subsequently, 10 μL of CCK-8 reagent was added to each well, and the plates were incubated for an additional 30 min. Absorbance was measured at 450 nm using a microplate reader (Sunrise™, Tecan, Switzerland). Cell viability was expressed as a percentage relative to untreated control cells.

2.9. In vivo imaging and acute safety of LNPs

2.9.1. In vivo bioluminescence imaging

To confirm successful in vivo mRNA delivery and expression prior to assessing adaptive immunity, bioluminescence imaging was performed. Mice were intramuscularly injected with LNPs encapsulating firefly luciferase mRNA (5 μg per mouse). At 6 h post-injection, D-luciferin potassium salt (3 mg per mouse) was administered intraperitoneally. After 10 min, bioluminescence signals were captured using an IVIS Lumina imaging system (PerkinElmer, USA). Signal intensity was quantified as average radiance (photons/s/cm2/sr) within defined regions of interest (ROIs) covering the injection site and major organs using Living Image software (PerkinElmer).

Assessment of the acute safety profile of LNPs: A separate cohort of mice was re-immunized with 10% TPS and 20% TPS modified LNP formulations (empty or luciferase mRNA-loaded, 5 μg mRNA per mouse, n = 6). Blood samples were collected via retro-orbital bleeding at 6 and 24 h post-immunization for plasma and serum separation. For plasma collection, blood was transferred to EDTA-coated tubes and centrifuged at 2000 × g for 15 min at 4 °C. For serum collection, blood samples were placed in a 37 °C incubator for 1 h to allow clot formation, then transferred to 4 °C overnight. After overnight storage, the samples were centrifuged at 4500 rpm for 30 min at 4 °C. Levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured using a Hitachi 7180 clinical analyzer (Hitachi High-Tech Corporation, Tokyo, Japan). IL-1β was detected using a commercial enzyme-linked immunosorbent assay (ELISA) kit (Invitrogen, #BMS6002–2) according to the manufacturer's protocol.

2.9.2. Assessment of serum cytokine profile

The anti-inflammatory properties of TPS-modified LNPs were evaluated through systematic cytokine profiling. Mice were administered a single intramuscular injection of one of seven formulations (n = 5 per group): PBS (vehicle control); empty LNPs (SM-102, 10% TPS, or 20% TPS); or corresponding RSV mRNA-encapsulated LNPs (standardized to 5 μg mRNA). To capture both the acute response and resolution phases, blood samples were collected via retro-orbital blood collection at 6 and 24 h after injection. Serum was separated by centrifugation and stored at −80 °C. Concentrations of IL-6 (a major pro-inflammatory mediator) and IFN-β (a key cytokine in nucleic acid-sensing pathways) were quantified using commercial enzyme-linked immunosorbent assay (ELISA) kits specific for mouse cytokines (IL-6: Invitrogen, #KMC0061; IFN-β: BioLegend, #439407) according to the manufacturers' protocols.

2.9.3. Vaccination schedule

Female BALB/c mice (6–8 weeks old, n = 5 per group) were randomly divided into 12 groups. Each mouse received an intramuscular injection of 125 μL vaccine formulation containing 5 μg of RSV mRNA into the medial thigh muscle. A standard SM-102-based LNP vaccine served as the positive control, while PBS acted as the negative control. Mice were immunized on days 0 and 14, and serum samples were collected on days 10 and 24 post-primary immunization for analysis of IgG, IgG1, and IgG2a antibody titers. On day 27 post-initial immunization, mice were euthanized, and spleens were aseptically harvested for evaluation of T cell-mediated immune responses and T cell memory phenotypes.

2.9.4. Analysis of antigen-specific antibody responses

Blood samples were collected via retro-orbital sinus on days 10 and 24 after the primary immunization. Serum was separated by centrifugation and stored at −80 °C. RSV pre-F protein-specific antibody titers (total IgG, IgG1, and IgG2a) were determined by enzyme-linked immunosorbent assay (ELISA). Briefly, 96-well plates were coated with RSV pre-F protein (100 ng/well). After blocking, serially diluted serum samples were added, followed by horseradish peroxidase (HRP)-conjugated anti-mouse IgG, IgG1, or IgG2a secondary antibodies (SouthernBiotech, USA). The reaction was developed with TMB substrate, stopped, and the absorbance was measured at 450/630 nm. Endpoint titers were defined as the highest serum dilution yielding an optical density greater than 0.105.

2.9.5. Live-virus neutralization assay

The functional capacity of serum antibodies was assessed by a microneutralization assay. Heat-inactivated serum samples were serially diluted and incubated with the RSV A2 strain (2000 TCID50/mL) for 2 h at 4 °C. The mixture was then transferred to a monolayer of HEp-2 cells and incubated for 3 days. RSV infection was detected by immunofluorescence using a rabbit anti-RSV F monoclonal antibody and an Alexa Fluor® 488-conjugated secondary antibody. The neutralizing antibody titer was defined as the highest serum dilution that reduced the number of fluorescent foci by 50% compared to virus control wells.

2.9.6. Analysis of antigen-specific T cell responses

On day 27, spleens were aseptically harvested. A single-cell suspension of splenocytes was prepared by mechanical dissociation and density gradient centrifugation. IFN-γ and IL-17 A FluoroSpot assay: To quantify antigen-specific T cells, splenocytes (2.5 × 105 cells/well) were stimulated with an overlapping peptide pool (length = 15 aa, overlap = 10 aa) spanning the RSV pre-F protein (2 μg/mL per peptide) in pre-coated IFN-γ and IL-17 A FluoroSpot plates (Mabtech, Sweden). After 36–48 h of incubation, spots were developed according to the manufacturer's protocol and counted using an automated reader (S6 Universal M2, CTL).

Flow cytometric analysis of memory T cells: Splenocytes were stained with a cocktail of fluorescently conjugated antibodies against CD3, CD4, CD8, CD44, CD62L, and CD11a, as detailed in Appendix A. Naïve (CD11alo CD44lo), central memory (TCM, CD62L+ CD44hi), and effector memory (TEM, CD62L− CD44hi) T cell populations within CD4+ and CD8+ subsets were identified and quantified by flow cytometry (CytoFLEX LX, Beckman Coulter; Fig. S1).

2.9.7. Flow cytometry analysis of antigen-presenting cells (APCs) in draining lymph node

A separate cohort of BALB/c mice received a single intramuscular injection of 5 μg RSV mRNA encapsulated in Std LNPs, TPS-modified LNPs, or PBS as a control. At 24 h post-immunization, mice were euthanized and inguinal draining lymph nodes were harvested and processed into single-cell suspensions by mechanical dissociation. Single-cell suspensions were blocked with anti-CD16/CD32 antibody and stained with a multi-color antibody panel. After viability staining, cells were analyzed on a CytoFLEX LX flow cytometer. Dendritic cell subsets (cDC1, cDC2, pDC), monocytes (classical and non-classical), macrophages and B cells were identified based on established surface marker combinations, as detailed in Appendix A. Activation status was assessed by quantifying the geometric mean fluorescence intensity (gMFI) of CD40, CD86, MHC-II, and CCR7 (on DCs only) within each defined population using FlowJo software (Fig. S2).

2.9.8. Evaluation of systemic safety and anti-inflammatory profile in vivo

To assess the safety of the modified LNPs-RSV mRNA vaccine, mice were immunized with LNPs-mRNA formulations containing 10% TP, 10% TPS, or 100% TPGS. Throughout the immunization period, the general health status of the mice was closely monitored, and their body weights were recorded every two days. On day 27 post-immunization, serum samples, and tissues from the liver and kidneys were collected for further analysis of biochemical parameters and histopathological evaluation. The tissues were fixed in 4% paraformaldehyde, cleared with xylene, embedded in paraffin wax, and sectioned for hematoxylin and eosin (H&E) staining to evaluate histopathological changes.

2.10. Statistical analysis

Data are presented as mean ± standard error of the mean (SEM). Statistical analysis was performed using GraphPad Prism version 10.1.2. For all analyses, the standard LNP formulation (Std-LNP) served as the primary reference group. Differences between multiple groups were analyzed either by one-way ANOVA followed by Tukey's or Dunnett's multiple comparisons test, or by two-way ANOVA followed by Šídák's multiple comparisons test, as appropriate for the experimental design. Statistical significance is indicated as: ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

3. Results and discussion

3.1. Rational design and physicochemical characterization of LNPs

Modified lipid nanoparticles were prepared by incorporating varying mass ratios of α-tocopherol and its derivatives into a conventional SM-102-based formulation (Fig. 1A-B). Given that the formulation process may affect the final incorporation efficiency, we next quantified the actual TPS content in the 10% and 20% TPS-modified LNPs using HPLC-CAD. As summarized in Table S1-S2 and illustrated in Fig. S3, the measured TPS content was approximately 50% of the theoretical input for both formulations (10% TPS: 41%; 20% TPS: 53.7%). Importantly, the relative proportions of the other four lipid components (SM-102, DSPC, cholesterol, and DMG-PEG2000) remained consistent with the intended formulation ratios, indicating that the preparation process did not alter the overall lipid composition beyond the reduced TPS incorporation. For clarity and consistency with the formulation design, all groups are nevertheless named according to their theoretical incorporation percentages.

The physicochemical properties of the resulting nanoparticles, including particle size, zeta potential, and encapsulation efficiency, were systematically evaluated to assess the impact of the incorporated components on nanoparticle characteristics. As shown in Fig. 1C, the particle size of the modified nanoparticles ranged from 90 to 120 nm, with all polydispersity index (PDI) values below 0.2. Compared to the standard SM-102 LNPs, the modified nanoparticles exhibited comparable particle sizes and PDI values, indicating that the incorporation of α-tocopherol and its derivatives did not cause significant alterations. For the three different derivatives, the trends in particle size and PDI varied slightly depending on the incorporation ratio. For TP, a 10% incorporation ratio resulted in larger particle sizes compared to the other two ratios. In contrast, for TPS, the particle size decreased gradually as the incorporation ratio increased. For TPGS within the 5–20% range, particle size followed a trend similar to TPS; however, complete replacement of DMG-PEG2000 with TPGS resulted in a significant size increase to approximately 120 nm. The PDI generally decreased with higher incorporation of TP or TPS but showed an opposite, increasing trend for TPGS.

Zeta-potential measurements revealed a weakly negative surface charge (−6 to 0 mV) for all formulations (Fig. 1C). The negative surface charge of LNPs may reduce non-specific interactions with plasma proteins and minimize perturbation of cell membranes, thereby enhancing both delivery efficiency and biocompatibility (Kong et al., 2024). Encapsulation efficiency is widely acknowledged as a key parameter for assessing the ability of LNPs to effectively encapsulate and retain therapeutic agents, which directly correlates with formulation quality and delivery performance (Ma et al., 2025). Following modification with varying ratios of TP and TPS, the encapsulation efficiency remained consistently above 90%, indicating that these formulations maintained high mRNA-loading capabilities (Fig. 1D). However, for TPGS-modified LNPs, a gradual decline in encapsulation efficiency was observed as the TPGS content increased. At a TPGS ratio of 20%, the encapsulation efficiency dropped to approximately 75%, suggesting that excessive TPGS incorporation may interfere with the formation or stability of the lipid core, consequently affecting mRNA retention.

3.2. Enhance mRNA delivery efficiency while maintaining biocompatibility in vitro by modified LNPs

The delivery efficiency of modified LNPs was initially evaluated in vitro at the cellular level. To ensure the accuracy and generalizability of the results, two different mRNA constructs, which encode luciferase and enhanced green fluorescent protein (eGFP), were used as model transcripts. As illustrated in Fig. 2A, all modified formulations efficiently delivered both mRNAs into cells, resulting in robust protein expression. Especially, when delivering eGFP mRNA, LNPs modified with 10% and 20% TPS demonstrated significantly increased GFP expression compared to the standard formulation group, with enhancements of 4.1- and 9.5-fold in protein levels, respectively. Conversely, no statistically significant differences were observed in GFP expression between the other modified formulations and the control group. For luciferase mRNA, relative to the SM-102 control formulation, the incorporation of 5% TP, 5% TPS, and 10% TPS markedly improved luciferase expression, resulting in 1.3-, 1.5-, and 1.8-fold increases, respectively. In contrast, formulations containing 10% TP, 20% TP, or 5% TPGS did not show statistically significant differences from the control. These findings suggest that low-to-moderate concentrations of these additives can enhance transfection performance. However, further increasing the concentration of TPS or TPGS (e.g., 20% TPS, 10% TPGS, 20% TPGS, or even 100% TPGS) resulted in a notable reduction in luciferase expression. Collectively, the overall transfection results suggest that the incorporation of TPS positively affects transfection efficiency, while other components may provide limited enhancement and, in some cases, lead to a decrease in transfection efficiency due to variations in their formulation ratios.

Fig. 2.

Fig. 2

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In vitro transfection and cellular uptake of mRNA delivered by different lipid nanoparticles. (A) eGFP and Luciferase expression caused by multiple LNP-mRNA formulations measured in HEK-293 T cells. (B) Representative confocal microscopy images of HEK-293 T cells after 4 h incubation with DiD-labeled LNPs (red). Nuclei were stained with DAPI (blue). Scale bar: 20 μm. (C) Quantitative analysis of integrated density was performed using ImageJ software by outlining individual cells in the bright-field image and measuring the corresponding fluorescence intensity in the DiD channel. (D) Cell viability of HEK-293 T cells treated with various formulations (CCK-8 assay). (E, F) Effect of endocytic pathway inhibitors on cellular uptake of 20% TPS-modified LNPs. Cells were pretreated with various inhibitors targeting specific uptake pathways: amiloride (macropinocytosis, 50 μM), chlorpromazine and sucrose (clathrin-mediated endocytosis; 15 μg/mL; 0.5 mM), Mβ-CD (lipid raft/caveolae-mediated endocytosis, 4 mM). Cellular uptake was quantified by flow cytometry and presented as mean fluorescence intensity (MFI). Data are presented as mean ± SEM. Statistical analyses were performed using GraphPad Prism version 10.1.2. Different statistical approaches were applied as appropriate: in (A) and (D), differences were analyzed by one-way ANOVA followed by Dunnetts multiple comparisons test, with comparisons made against the Std-LNP group (A) or the PBS control group (D). In (C), differences among all groups were analyzed by one-way ANOVA followed by Tukeys multiple comparisons test for all pairwise comparisons. In (E, F), differences among all groups were analyzed by two-way ANOVA followed by Šídáks multiple comparisons test for all pairwise comparisons. Statistical significance is indicated as: ns, not significant, not identified in figure; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

TP and its derivatives are reported to promote nanoparticle internalization via multiple endocytic pathways, including receptor-mediated uptake through scavenger receptor class B type I (SR-BI) and Niemann-Pick C1-like 1 (NPC1L1), as well as lipid-raft/caveolin-dependent internalization (Takada and Suzuki, 2010; Cui et al., 2025). To assess whether enhanced cellular entry contributes to the superior transfection efficiency of modified LNPs, we first visualized their uptake using confocal microscopy. HEK-293 T cells were incubated with DiD-labeled LNPs for 4 h. As shown in Fig. 2B, all LNP formulations were successfully internalized. Notably, cells treated with the 20% TPS-modified LNPs exhibited markedly more intense intracellular fluorescence compared to those treated with the standard SM-102 LNPs, suggesting enhanced cellular uptake or accumulation of the nanoparticles. In contrast, TPGS-modified formulations showed fluorescence intensity similar to the control (Fig. 2C). The notably stronger signal from the 20% TPS group aligns with its highest transfection efficiency, supporting the inference that improved cellular uptake may be a key factor driving the enhanced protein expression.

To extend these observations, we next assessed the cellular internalization of the 20% TPS-LNP formulation in HepG2 cells. Classical endocytosis inhibitors were subsequently employed to delineate the uptake mechanisms in both HEK-293 T and HepG2 cells, permitting a direct comparison of internalization routes utilized by TPS-LNPs versus unmodified LNPs. Across both cell lines, TPS-LNPs consistently demonstrated superior internalization efficiency relative to their counterparts. Treatment with a panel of four distinct inhibitors revealed a congruent inhibitory profile for both formulations. Notably, chlorpromazine, an inhibitor of clathrin-mediated endocytosis, elicited the most pronounced suppression of uptake, confirming that clathrin-dependent endocytosis constitutes the principal entry pathway for both LNP types (Fig. 2E- F). Given that the mechanistic signature of inhibition remained unchanged while absolute uptake was markedly augmented, we postulate that the enhanced cellular entry of TPS-LNPs stems from alterations in interfacial biophysical properties rather than a diversion to distinct or parallel uptake mechanisms, as previously identified by Dudek et al. (Dudek et al., 2025).

The potential side effects of LNPs have consistently been a critical concern in the development and application of lipid nanoparticle-based delivery systems. Therefore, the cytotoxicity of the modified LNP formulations was assessed at the cellular level using standard cell viability assays. As shown in Fig. 2D, all modified LNP formulations exhibited high cell viability, with survival rates exceeding 90%. No significant differences were observed compared to the positive control (SM-102) and the PBS groups. These findings indicate that the inclusion of TP and its derivatives does not compromise cellular health under the tested conditions, highlighting their potential for safe therapeutic applications. Further investigations will focus on evaluating their acute and systemic inflammatory responses in vivo.

3.3. Enhances mRNA delivery in vivo and attenuates LNP-induced systemic inflammatory cytokine release by modified LNPs

The in vitro studies demonstrated that modified lipid nanoparticles significantly enhanced both mRNA expression and cellular uptake efficiency, with minimal cytotoxicity observed across all tested formulations. These findings suggest a promising profile for in vivo translation. To investigate the feasibility of systemic delivery, we conducted small-animal live imaging following intramuscular administration of LNPs encapsulating luciferase mRNA. This approach enabled real-time visualization and quantitative assessment of luciferase mRNA expression in vivo, providing critical insights into the in vivo delivery performance of these formulations. As depicted in Fig. 3A-B and Fig. S4, luciferase expression was detected in all groups following intramuscular administration. The 10% TPS formulation consistently showed the highest total bioluminescent signal among all tested groups, though this increase did not reach statistical significance compared to the standard SM-102 LNPs. Notably, the 20% TPS, 5% TP, and 10% TPGS formulations all achieved expression levels comparable to the SM-102 control (Fig. 3B). Several other modified formulations exhibited reduced systemic expression. These results demonstrate that specific α-tocopherol modifications-particularly TPS at both 10% and 20% incorporation-can achieve in vivo delivery efficacy that matches or potentially exceeds conventional LNP benchmarks, with 10% TPS showing a particularly promising trend toward enhanced performance. However, in vivo imaging at the 6-h time point also revealed substantial hepatic accumulation of both standard SM-102 LNPs and TPS-modified LNPs, accounting for approximately one-third to one-half of the total bioluminescent signal. To investigate whether this liver sequestration could induce acute hepatotoxicity, we performed liver function tests at the same 6-h time point and again at 24 h post-administration. We measured plasma levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), two well-established biochemical markers of liver function and injury. The results (Fig. 3C, Fig. S5) showed that, compared with the PBS control group, neither the Std-LNPs nor the TPS-LNPs groups exhibited abnormal elevations of these markers, indicating that these LNP formulations did not cause acute liver injury.

Fig. 3.

Fig. 3

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In vivo performance and assessment of acute safety profile of modified LNPs. (A) Bioluminescent images of mice 6 h after intramuscular (i.m.) administration of TPS-modified, luciferase mRNA-loaded LNPs (5 μg mRNA per mouse, n = 3 per group). (B) Quantitative analysis of total flux (radiance sum from region of interest) derived from images in (A). (C) Plasma biochemistry analysis of ALT and AST. Levels were measured at 6 h and 24 h after i.m. administration of mRNA-loaded LNPs (5 μg luciferase mRNA per mouse, n = 6 per group). (D, E) Serum concentrations of (D) IL-6 and (E) IFN-β. Levels were measured at 6 h and 24 h after i.m. administration of either blank LNPs (without mRNA) or RSV mRNA-loaded LNPs (5 μg mRNA per mouse). All formulations in (D) and (E) correspond to the Std-LNP, 10% TPS, and 20% TPS groups. Data are presented as mean ± SEM. Statistical analyses were performed using GraphPad Prism version 10.1.2. Differences in (B) were analyzed by two-way ANOVA (factors: formulation and region) followed by Šidák's multiple comparisons test, with all comparisons made against the Std-LNP group at the corresponding expression region. Differences in (C), (D) and (E) were analyzed by one-way ANOVA followed by Tukey's multiple comparisons test for all pairwise comparisons within each time point. Statistical significance is indicated as: ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

We next assessed whether the superior delivery efficiency of TPS-modified LNPs was accompanied by a favorable safety profile through systematic cytokine profiling. Serum levels of IL-6, IFN-β and IL-1β were quantified at 6 and 24 h after immunization with SM-102, 10% TPS, and 20% TPS LNPs, each in both mRNA-loaded and empty forms. For the pro-inflammatory cytokine IL-6, TPS modification markedly attenuated the acute response induced by the nanoparticle carrier itself. At 6 h post-injection of empty LNPs, serum IL-6 levels in the SM-102 group were significantly higher than those in both the 10% TPS and 20% TPS groups, which themselves showed no significant difference from each other. By 24 h, IL-6 concentrations had declined in all groups, with levels in the SM-102 group remaining numerically higher than in the TPS-modified groups, though the difference was no longer statistically significant. A similar protective effect was observed when LNPs were loaded with mRNA. Following administration of mRNA-encapsulated formulations, the 6-h IL-6 response again showed a significant reduction in both TPS-modified groups compared to the SM-102 control. At the 24-h time point, all groups exhibited resolved cytokine levels, with no statistically significant differences among them; however, the 20% TPS group consistently showed the lowest numerical value (Fig. 3D). We speculate that the significant decrease in IL-6 levels caused by TPS incorporation may be attributed to two possible mechanisms. First, TP and its derivatives are well-recognized for their antioxidant properties. TPS-modified LNPs may inhibit the activation of NF-κB through reducing oxidative stress (Sharma and Vinayak, 2011; Yu et al., 2025). Second, IL-6 signal transduction depends on the assembly of its receptor subunits (IL-6Rα and gp130) within cholesterol-rich lipid raft microdomains, which facilitates STAT3 activation. TPS incorporation into LNPs might alter membrane raft composition or fluidity, thereby interfering with IL-6 receptor clustering and downstream signaling, which could feedback to reduce sustained IL-6 production (Woo et al., 2022; Yu et al., 2023). These mechanisms, along with other possible pathways, likely act in concert to lower IL-6 levels. However, dedicated experimental studies are needed to verify these hypotheses.

IFN-β responses revealed a distinct, context-dependent immunomodulatory profile for TPS modification. Following administration of empty LNPs, all groups maintained low IFN-β levels at both time points. At 6 h, concentrations in the SM-102 group were numerically lower than in the 10% and 20% TPS groups, with the latter showing the highest value, although these differences did not reach statistical significance. By 24 h, IFN-β levels had further declined to near-baseline in all groups. In stark contrast, when LNPs were loaded with mRNA, the IFN-β response pattern was reversed. At 6 h post-injection, the SM-102 group exhibited substantially higher serum IFN-β levels than both TPS-modified groups (Fig. 3E). This elevation persisted at 24 h, with SM-102 levels remaining numerically elevated above those of the TPS groups, even as overall cytokine concentrations declined. This bidirectional regulation of IFN-β underscores a sophisticated, “calibrating” function for TPS within the LNP. Rather than acting as a blanket immunosuppressant, TPS appears to dynamically modulate the nucleic acid-sensing pathway based on the immune context. The suppression of excessive type-I interferon signaling in the presence of mRNA is critically advantageous. Beyond mitigating a key mediator of systemic reactogenicity, it may prevent the translational inhibition and immune dysregulation often associated with strong IFN-β responses, thereby enhancing the quality and magnitude of the adaptive immunity (Lobb et al., 2026; Palacio et al., 2020). Given the well-documented pro-inflammatory properties of conventional ionizable cationic lipids, which can activate the NLRP3 inflammasome and trigger IL-1β mediated reactions (Moghimi and Simberg, 2022; Omo-Lamai et al., 2025), we also quantified serum IL-1β levels. The results showed that IL-1β levels in all LNP-treated groups (Std, 10% TPS, 20% TPS) remained low at both 6 and 24 h post-injection and were not significantly different from the PBS control group (Fig. S6). This confirmed our hypothesis that, under the tested conditions, these LNP formulations do not promote the maturation and secretion of IL-1β. Collectively, these findings elevate α-tocopherol succinate from a mere structural lipid to an active immunomodulatory component that simultaneously enhances safety and may favorably shape the efficacy of mRNA-LNP vaccines.

3.4. In vivo immune responses elicited by modified LNP-encapsulated RSV mRNA vaccine

To evaluate the immunogenic potency of the engineered LNPs, we employed an RSV mRNA vaccine model in mice immunized on days 0 and 14 (Fig. 4A). All modified LNPs elicited robust antigen-specific humoral responses (Fig. 4B-4D). After prime immunization, serum anti-RSV pre-F IgG titers reached up to 104, an effect largely comparable to the SM-102 control across most formulations. A booster immunization significantly amplified antibody production, with peak titers exceeding 105 (Fig. 4B, Fig. S7). Throughout the immunization schedule, the 10% TPS formulation consistently induced antibody levels on par with the SM-102 benchmark, whereas the 20% TP and 100% TPGS groups elicited relatively lower responses (Fig. 4B, Fig. S7). To assess the functional quality of the humoral response, we measured serum neutralizing antibody (nAb) titers against live RSV. All LNP formulations induced nAb titers statistically comparable to the SM-102 control (Fig. S7). Notably, however, the 10% TP and 10% TPS groups exhibited notably elevated mean nAb titers 1.9-fold and 1.6-fold higher than the SM-102 group (Fig. 4D, Fig. S7), respectively, suggesting a trend toward enhanced functional immunogenicity conferred by these specific modifications.

Fig. 4.

Fig. 4

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Humoral and cellular immunogenicity. (A) Timeline of vaccination and sample collection. (B) The RSV pre-F protein-specific IgG antibody levels of modified LNP-mRNA complexes after the boost immunization, as detected by ELISA. (C) The RSV pre-F protein-specific IgG1 and IgG2a antibody levels and the corresponding ratio of LogIgG2a/LogIgG1 of modified LNP-mRNA complexes after the boost immunization, as detected by ELISA. (D) The neutralizing antibody titers against live virus across all treatment groups. (E) The quantification of restimulated IFN-γ-secreting and IL-17 A-secreting splenocytes was verified in the FluoroSpot test. (F) Results of TEM and TCM in splenic CD4+ T cells. (G) The percentage of naïve CD4+ T cells and CD8+ T cells. (H) Results of TEM and TCM in splenic CD8+ T cells. Data are presented as mean ± SEM. Statistical significance was determined using one-way ANOVA followed by Dunnett's multiple comparisons test, with all comparisons made against the Std-LNP group. All data showed no statistically significant differences and were not labeled in the figure.

mRNA vaccines are particularly notable for their ability to elicit strong T-cell-mediated immunity, a feature that distinguishes them from conventional platforms, such as inactivated and subunit vaccines (Ura et al., 2022). Therefore, antigen-specific cellular immune responses and memory T cells of all modified LNPs-RSV mRNA vaccines were evaluated. FluoroSpot assays revealed that all LNP formulations stimulated interferon-γ (IFN-γ) secretion, with the 20% TPS and 100% TPGS groups showing a notable increase-mean responses were approximately 1.4-fold higher than the SM-102 control, indicating enhanced T cell immunostimulation. In parallel, analysis of IL-17 A, a cytokine linked to Th17-polarized inflammatory responses, revealed a distinct pattern: except for the 5% TP group, all other modified formulations induced lower mean IL-17 A levels relative to the SM-102 control (Fig. 4E, Fig. S8). This coordinated enhancement of IFN-γ together with a moderation of IL-17 A suggests that TPS and TPGS modifications not only amplify Th1-driven protective immunity but may also actively temper Th17-associated inflammatory pathways, thereby promoting a more balanced and potentially safer T-cell response profile. Flow cytometry analysis of T cell populations revealed that the modified LNP formulations led to expanded proportions of memory CD8+ T cells (Fig. S8). Notably, the 20% TPS-modified LNPs significantly increased the number of effector memory CD8+ T cells (Fig. 4H), corresponding with a decrease in the proportion of naïve CD8+ T cells (Fig. 4G). Together, these findings indicate that TPS modification enhances both the magnitude and quality of antigen-specific cellular immunity, favoring the development of a differentiated and potentially more protective CD8+ T cell response.

3.5. Immunoenhancing mechanisms of TPS-modified LNPs-mRNA vaccine

To elucidate the cellular mechanisms underlying the enhanced immunogenicity of TPS-modified LNPs, we performed a comprehensive immunophenotypic analysis of antigen-presenting cells (APCs) in draining lymph nodes 24 h post-immunization. The 10% TPS formulation induced a distinct APC activation profile characterized by a targeted upregulation of the key co-stimulatory molecule CD40. Compared to the SM-102 control, TPS-modified LNPs elicited significantly higher CD40 expression on monocytes and macrophages (Fig. 5A). This enhancement was specific, as other functional markers (e.g., MHC-II, CD86) generally remained comparable between the two LNP groups across most APC subsets (Fig. 5C, Fig. S9–10). A notable shift in monocyte polarization was also observed: the TPS group exhibited a significantly lower ratio of classical to non-classical monocytes than the SM-102 group (Fig. 5B), suggesting a modulation of the innate immune milieu toward a less inflammatory state. Collectively, these data highlight that TPS modification does not broadly amplify APC activation, but rather recalibrates it. The pronounced upregulation of CD40 specifically on monocytes and macrophages provides a direct mechanistic bridge between innate sensing and adaptive immunity. CD40-CD40L signaling is known to license antigen-presenting cells for robust T cell priming and skew toward Th1 responses (Bullock, 2022; Tang et al., 2021), which aligns with our observed enhancement in IFN-γ+ T cells and neutralizing antibodies. Importantly, this immunomodulation is context-dependent: while CD40 is upregulated, other activation markers (MHC-II, CD86) remain stable, and inflammatory cytokines (IL-6) are subdued. This “calibrated” activation profile epitomizes the dual-functionality of TPS-LNPs: promoting productive immunity without eliciting excessive inflammation.

Fig. 5.

Fig. 5

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Flow cytometry analysis of functional markers of DCs, monocytes, macrophages and B cells in the draining lymph node. (A) Frequency and CD40 expression of DCs, monocytes, and macrophages at 24 h post-immunization. (B) The ratio of classical to non-classical monocytes. (C) Heatmap analysis of functional markers expression. CD86, cluster of differentiation 86; CD40, cluster of differentiation 40; MHC-II, major histocompatibility complex class II; CCR7, C-C chemokine receptor type 7. Data are presented as mean ± SEM. Statistical significance was determined using one-way ANOVA followed by Tukey's multiple comparisons test, and results are indicated as follows: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

3.6. In vivo safety evaluation of modified LNP-encapsulated RSV mRNA vaccine

To comprehensively evaluate the in vivo safety profile of the modified LNPs, we performed a multi-parametric assessment that included longitudinal body-weight monitoring, serum biochemistry, and histopathological analysis. The following groups were examined: PBS (vehicle control), Std-LNP, 10% TP-LNPs, 10% TPS-LNPs, and 100% TPGS-LNPs. Mice (n = 3 per group) were immunized as outlined in the “Evaluation of systemic safety and anti-inflammatory profile in vivo” protocol. Throughout the 27-day study, all vaccinated groups maintained normal weight gain (Fig. 6A). At the end of the observation period, the mice were euthanized, and samples of serum, liver, spleen, and kidney tissues were collected for analysis. Organ function safety was evaluated by measuring serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), urea (UREA), and uric acid (UA), as well as by hematoxylin and eosin (H&E) staining. As shown in the data, the levels of ALT, AST, and ALP across all groups remained within normal physiological ranges, indicating no acute liver injury or cholestasis (Fig. 6B). Urea and uric acid levels were also stable, demonstrating that the vaccine components did not affect glomerular filtration or uric acid metabolism (Fig. 6C). Furthermore, the histopathological examination confirmed that the morphological structures of liver, spleen, and kidney tissues in all groups remained intact. No significant pathological alterations, such as inflammation, necrosis, or fibrosis, were observed (Fig. 6D).

Fig. 6.

Fig. 6

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In vivo long-term safety evaluation. (A) Changes in the mice's body weight during treatment time. (B, C) The liver and kidney function of mice in each group after two immunizations as determined by blood biochemical indexes (n = 3). ALT, AST and ALP represented liver function, while UREA and UA represented kidney function. Data are presented as mean ± SEM. Statistical significance was determined using one-way ANOVA followed by Dunnett's multiple comparisons test, with all comparisons made against the PBS group. All data showed no statistically significant differences and were not labeled in the figure. D) Representative histopathology (H&E) of liver and kidney in different groups (scale bar = 100 μm). H&E-stained sections shown in the data were one representative result of the corresponding three tested mice.

4. Conclusion

In summary, this study establishes a novel design paradigm for LNP-based mRNA delivery by leveraging the intrinsic dual functionalities of α-tocopherol (TP) derivatives. We hypothesized that integrating these molecules directly into the LNP matrix could simultaneously mitigate carrier-induced reactogenicity and enhance antigen-specific immunogenicity, a long-standing challenge in the field. Through rational design and systematic evaluation, we have validated this hypothesis and elucidated the underlying mechanisms. Our key findings are threefold.

First, at the formulation level, we successfully confirmed TPS incorporation into LNPs by HPLC-CAD. Despite the measured TPS content being approximately 50% of the theoretical input, the modified LNPs retained favorable physicochemical properties and, importantly, exhibited significantly enhanced cellular uptake across two different cell lines, which translated into superior in vitro and in vivo mRNA delivery efficiency without compromising biocompatibility. Second, regarding safety, TPS modification effectively attenuated acute systemic inflammation, as evidenced by markedly reduced levels of pro-inflammatory cytokines (IL-6 and IL-1β) post-immunization, while maintaining a benign systemic safety profile in comprehensive toxicological assessments. Third, and most importantly, at the immunological level, TPS-LNPs elicited a robust and balanced adaptive immune response. This was characterized by potent and functional humoral immunity (high-titer neutralizing antibodies) and enhanced cellular immunity, including a skew toward Th1-type responses and the expansion of antigen-specific memory CD8+ T cells. Mechanistically, these benefits were linked to a calibrated activation of antigen-presenting cells, specifically the upregulation of the key co-stimulatory molecule CD40 on monocytes and macrophages, which promotes productive T cell priming without unleashing generalized inflammatory cascades.

Collectively, this work transcends the conventional view of excipients by repositioning α-tocopherol derivatives as active immunomodulatory components within the LNP architecture. We demonstrate that they function not merely as structural lipids or antioxidants, but also as intrinsic biological modifiers capable of “recalibrating” the host's immune response to the delivery vector and its cargo. This approach successfully decouples the traditional efficacy-safety trade-off, offering a versatile and generalizable platform strategy. While this study provides a proof-of-concept in a vaccine model, several avenues remain for future exploration. The structure-activity relationship of other TP derivatives, the long-term durability of immune responses and safety, and the platform's applicability to therapeutic mRNA delivery beyond vaccines (e.g., protein replacement, gene editing) warrant further investigation. Additionally, leveraging this design principle for targeted delivery to extrahepatic tissues represents a promising direction for broadening the therapeutic scope of mRNA-LNP technology. Ultimately, by integrating multifunctional biomolecules with inherent biological activity into nanocarrier design, we pave the way for the next generation of “smarter” delivery systems that are not only efficient but also intelligently modulate their biological interfaces for optimal therapeutic outcomes.

Funding

This work was supported by National Key Research and Development Program (2025YFC2311703) and National Vaccine and Serum Institute (KTZC1900918A).

CRediT authorship contribution statement

Huijun Wang: Writing – original draft, Validation, Investigation. Ziyao Kang: Validation, Methodology, Investigation, Formal analysis. Xiaoshuang Wang: Validation, Investigation, Formal analysis. Zheng Pan: Validation, Investigation. Yun Sun: Writing – review & editing, Visualization, Formal analysis. Yuan Li: Validation, Investigation. Xin Li: Validation, Investigation. Shuai Shao: Validation, Investigation. Xiang Zheng: Validation, Investigation. Zibo Han: Writing – review & editing, Visualization, Resources, Methodology, Investigation, Formal analysis. Jin Ren: Writing – review & editing, Visualization, Supervision, Resources, Methodology, Funding acquisition, Formal analysis, Conceptualization.

Declaration of competing interest

The authors declare no competing financial interest.

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.ijpx.2026.100553.

Contributor Information

Zibo Han, Email: hanzibo@sinopharm.com.

Jin Ren, Email: renjin@sinopharm.com.

Appendix A. Supplementary data

Supplementary material

mmc1.docx (1.2MB, docx)

Data availability

Data will be made available on request.

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Associated Data

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Supplementary Materials

Supplementary material

mmc1.docx (1.2MB, docx)

Data Availability Statement

Data will be made available on request.

Articles from International Journal of Pharmaceutics: X are provided here courtesy of Elsevier

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