Covalent conjugation of TLR9 agonists to phospholipids enhances mRNA-LNP delivery efficiency and dendritic cell cross-priming

Abstract

Current mRNA-LNP systems face challenges in efficient co-delivery of nucleic acid adjuvants, limiting their capacity to elicit robust cellular immunity. To overcome the dissociation limitations of physically admixed CpG in conventional lipid nanoparticles, we developed a covalent conjugation platform that integrates TLR9 agonists with phospholipids via amide bonding. We synthesized a DSPE-CpG conjugate, which exhibited high loading efficiency (97.1%) onto mRNA-LNPs. The immune activation efficacy was evaluated in models encoding SARS-CoV-2 spike protein and HBV antigens. The DSPE-CpG-mRNA LNP system demonstrated enhanced cellular uptake via clathrin-mediated endocytosis, superior lymph node targeting of the adjuvant, and improved antigen expression in vivo. In the SARS-CoV-2 model, it induced high levels of IFN-γ⁺CD8⁺ T cells and elicited higher titers of specific IgG and neutralizing antibodies. In the HBV model, it significantly enhanced antigen-specific CD107a⁺ and FasL⁺ CTLs. Acute toxicity tests revealed no significant adverse effects. This covalent conjugation strategy, by ensuring co-localized delivery of antigen and adjuvant, provides an innovative and translatable platform for enhancing mRNA vaccine efficacy.

Graphical abstract

Supplementary Information

The online version contains supplementary material available at 10.1186/s12951-026-04394-2.

Introduction

The advent of mRNA-LNP technology has revolutionized nucleic acid delivery [1–3], yet efficient co-encapsulation of oligonucleotide adjuvants remains a critical barrier to achieving robust cellular immunity [4–6]. Conventional physical mixing of TLR9 agonists like CpG with mRNA-LNP suffers from adjuvant-antigen dissociation during lymphatic trafficking, leading to suboptimal dendritic cell (DC) uptake and compromised T cell responses [7–10]. While hydrophobic modification strategies (e.g., cholesterol conjugation) can enhance adjuvant stability, they often induce nanoparticle heterogeneity and unpredictable biodistribution due to lipid self-assembly tendencies [11, 12]—a fundamental nano-bio interface challenge demanding precise engineering solutions [12].

Recent advances reveal that covalent conjugation of nucleic acids to phospholipids offers superior control over nanostructure assembly [13, 14]. DSPE (1,2-distearoyl-sn-glycero-3-phospho-ethanolamine), a core component of clinical-grade LNPs [15], presents unique advantages: its C18 alkyl chains enable stable membrane integration, while the phosphoethanolamine headgroup permits site-specific amide bonding [16]. Critically, unlike cholesterol-modified constructs that cause phase separation [17], DSPE-based conjugates maintain nanoscale homogeneity and co-delivery fidelity—parameters essential for synchronized antigen/adjuvant uptake by DCs [18, 19].

Here, we bridge this gap through a structure-guided nanoengineering approach. By covalently conjugating CpG to DSPE via amide bonds, we constructed an integrated DSPE-CpG-mRNA LNP system. This design breaks through the limitations of existing technologies through a series of mechanisms: the lipid characteristics of DSPE enable its spontaneous combination with LNP, thereby forming a structurally stable CpG-mRNA co-delivery system; its amphiphilic structure enhances bio-membrane penetration [20], promoting synchronous uptake of both antigen and adjuvant by antigen-presenting cells (APCs) [21–23]; meanwhile, the lymph-targeting properties can optimize innate immune activation efficiency. We validated the efficacy of this platform in two distinct disease models—SARS-CoV-2 and HBV—demonstrating its ability to significantly enhance dendritic cell (DC) maturation and CD8 + T cell response intensity while maintaining excellent safety. This study provides a versatile nano-delivery strategy for nucleic acid therapeutics.

Methods

Cell lines and animals

Human embryonic kidney cells HEK-293 (ATCC^® CRL-1573^™), mouse C2C12 myoblasts cells (BeNa Culture Collection, Beijing, China), and immortalized dendritic cells DC2.4 (Shanghai Cell Bank, Chinese Academy of Sciences) were maintained in high-glucose DMEM (GNM12800, JiNuo Biotechnology Co. Ltd. Zhejiang, China) supplemented with 10% fetal bovine serum (FBS, 16140071, Gibco). Cells were cultured at 37 °C in a humidified atmosphere containing 5% CO₂ (Heraeus, Germany). Male BALB/c and C57BL/6 mice were purchased from Slaccas Experimental Animal Co., Ltd. (Shanghai, China) and bred under pathogen-free conditions. All animal experimental procedures were approved by the Institutional Animal Care and Use Committee of Zhejiang University (20241580).

DSPE CpG synthesis

CpG (5 mg, Xi′an ruixi Biological Technology Co.Ltd, CpG1826-5′-T*C*C*A*T*G*A*C*G*T*T*C*C*T*G*A*C*G*T*T-3′; CpG1018-5′-T*G*A*C*T*G*T*G*A*A*C*G*T*T*C*G*A*G*A*T*G*A-3′) was dissolved in 0.1 M carbonate buffer and mixed thoroughly. Pre-dissolved DSPE-NHS (10 equivalents in N, N-Dimethylformamide, DMF) was added to the solution. The reaction mixture was incubated overnight (16 h) at room temperature. The product was filtered and purified by High-Performance Liquid Chromatography (HPLC) using a C18 column (30 × 250 mm, 10 μm particle size). The mobile phases consisted of 0.1 M triethylamine acetate (TEAA, pH 7.0–7.1, phase A) and acetonitrile (ACN, phase B). The structural integrity of the purified DSPE-CpG conjugate was confirmed by mass spectrometry.

Preparation and characterization of DSPE CpG-mRNA LNP

SARS-CoV-2 RBD (amino acids 319–541) mRNA was synthesized by Ausper Biopharma Inc. (Hangzhou, China), based on a previously reported sequence [24]. mRNA-LNPs were prepared by microfluidic mixing. Briefly, a lipid mixture of DLin-MC3-DMA, DSPC (1,2-didecyl-sn-glycero-3-choline, from AVT Company), cholesterol, and DMG-PEG2000 (1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000, AVT) at a molar ratio of 50:10:38.5:1.5 was mixed with mRNA (mRNA/lipid weight ratio 1:25, N/P ratio 6) in 10 mM citrate buffer (pH 4.0) at a 1:3 volume ratio. The mixture was injected into a microfluidic device (ZX-LS-32, FluidicLab.) at a combined flow rate of 1.5 ml/min. The resulting LNPs were dialyzed against PBS (pH 7.4, 10 mM) for 16 h to remove ethanol. To prepare adjuvant-incorporated LNPs, DSPE-CpG or free CpG was added to pre-formed mRNA-LNPs at an mRNA: CpG mass ratio of 1:2 and incubated at 4 °C for 120 min. Particle size and polydispersity index (PDI) were measured by dynamic light scattering (DLS, Nano-ZS90, Malvern Zetasize). Morphology was observed by Cryo-electron microscopy (cryo-EM, Talos F200c, Thermo Fisher Scientific). mRNA encapsulation efficiency was determined using the Quant-iT RiboGreen RNA assay (RiboGreen RNA Quantitative Kit, P9745, Solarbio).

Cryo-electron microscopy imaging

The nanostructure of DSPE-CpG-mRNA LNP was analyzed using a 200 kV cryo-transmission electron microscope (Talos F200C, Thermo Fisher Scientific). Samples (3 µL of 0.1 mg/mL mRNA LNP) were applied to glow-discharged Quantifoil R2/2 holey carbon grids (300 mesh). Excess liquid was blotted under controlled conditions (blot force: 3, blot time: 5 s, wait time: 3 s, 100% humidity) using a Vitrobot Mark IV (Thermo Fisher Scientific) at 4 °C, followed by rapid plunging into liquid ethane cooled by liquid nitrogen. Grids were transferred to the microscope under cryogenic conditions. Images were acquired at a nominal magnification of 73,000×. Particle morphology analysis were performed using SerialEM for automated data collection.

Fluorescence quenching assay for surface accessibility

Surface localization of DSPE-CpG on LNPs was assessed using a membrane-impermeable quencher, potassium iodide (KI). Cy5-labeled DSPE-CpG was formulated as: (1) DSPE-CpG-mRNA-LNP, (2) core-encapsulated DSPE-CpG-mRNA-LNP, and (3) free Cy5-DSPE-CpG. Samples were diluted in PBS to equivalent Cy5 fluorescence. Initial fluorescence (F₀) was measured in a black 96-well plate (Ex/Em: 640/670 nm). KI quenching buffer (4.0 M KI) was added, incubated for 10 min at room temperature in dark, and fluorescence (F_KI) was recorded. Surface quenching efficiency was calculated as: QE (%) = (1 – F_KI/F₀) × 100. All measurements were performed in triplicate.

Extraction of bone marrow-derived dendritic cells (BMDCs)

Bone marrow cells were isolated from the tibiae and femurs of euthanized C57BL/6 mice (6–8 weeks old) by flushing with RPMI 1640 medium. Erythrocytes were lysed using pre-cooled red blood cell lysis buffer for 5 min. After centrifugation (1500 rpm, 5 min), cells were resuspended in BMDC culture medium: RPMI 1640 supplemented with 10% FBS, 10 ng/mL murine IL-4, and 20 ng/mL murine GM-CSF. Cells were seeded into 6-well plates at 2 × 10⁶ cells/mL (2 mL/well) and cultured. On day 2, half of the medium was replaced with fresh medium containing cytokines. Loosely adherent immature DCs (imDCs) were collected between days 6 and 7 for subsequent assays.

BMDC maturation in vitro

BMDCs were seeded in 24-well plates (5 × 10⁴ cells/well) and cultured overnight. Cells were treated with mRNA LNP, Free CpG-mRNA LNP, or DSPE-CpG-mRNA LNP (each containing 1.0 µg RBD mRNA) for 24 h. Cells were then collected, stained with PE-anti-CD11c, FITC-anti-CD80, and APC/Cyanine7-anti-CD86, washed twice with PBS, and analyzed by flow cytometry to assess maturation.

CpG loading efficiency determination

Cy5-labeled C18-CpG or DSPE-CpG was incubated with pre-formed mRNA-LNPs. Unbound conjugate was removed by ultrafiltration. Purified LNPs were lysed with 1% Triton X-100, and fluorescence intensity (Ex/Em: 640/670 nm) was measured. A standard curve was generated using known concentrations of Cy5-CpG. Loading efficiency was calculated as (mass of loaded CpG/total input CpG mass) × 100%.

In vitro transfection of mRNA LNP, Free CpG mRNA LNP and DSPE CpG mRNA LNP

DC2.4 cells, BMDCs, or HEK293T cells were seeded in 24-well plates. At about 70% confluence, medium was replaced with 450 µL serum-free medium. mRNA LNP, Free CpG-mRNA LNP, or DSPE-CpG-mRNA LNP was added to a final mRNA concentration of 1.0 µg/mL. After 4 h incubation, medium was replaced with complete medium containing 10% FBS. GFP expression was observed by fluorescence microscopy 20 h later.

Western blot

HEK293T cells were treated with mRNA LNP, Free CpG-mRNA LNP, or DSPE-CpG-mRNA LNP for 24 h. Cells were lysed in RIPA buffer containing protease and phosphatase inhibitors on ice for 10 min. Lysates were centrifuged (12,000 rpm, 15 min, 4 °C), and supernatant protein was denatured with 5× loading buffer. Proteins were separated by 10% SDS-PAGE, transferred to PVDF membranes, blocked with 5% skim milk, and incubated overnight at 4 °C with anti-GAPDH (1:1000) and anti-SARS-CoV-2 spike RBD (1:5000) antibodies. Membranes were washed with TBST, incubated with HRP-conjugated anti-rabbit IgG (1:5000) for 3 h at 37 °C, washed again, and developed using an ECL substrate. Band intensity was quantified with Image J.

Cellular uptake

HEK293T cells were seeded in 24-well plates. At ~60% confluence, DiD-labeled LNPs were added (final mRNA concentration 1 µg/mL). At indicated time points (0, 2, 8, 10, 24 h), cells were washed with PBS, stained with Hoechst 33342 (10 µg/mL, 20 min), washed again, and imaged by fluorescence microscopy. Images were analyzed with ImageJ.

Mechanisms of cellular internalization

BMDCs were seeded in 24-well plates (2.5 × 10⁵ cells/well) overnight. Cells were pre-treated for 30 min with endocytosis inhibitors: chlorpromazine (25 µM), nystatin (2.5 mg/mL), or amiloride (50 µM). Medium was replaced with inhibitor-containing medium with DiD-labeled LNPs (15 µg/mL mRNA) for 2 h. Cells were washed with PBS and imaged by fluorescence microscopy (DiD Ex: 644 nm). Data were analyzed with ImageJ.

Lysosomal escape

BMDCs were seeded on coverslips in 24-well plates (1 × 10⁵ cells/mL) overnight. Cells were treated for 12 h with DiD-labeled LNPs containing FITC-ssDNA (1.0 µg/mL). Lysosomes were stained with LysoTracker Red (50 nM, 30 min), nuclei with Hoechst 33342 (10 µg/mL, 15 min), and cells were fixed with 4% PFA. Coverslips were mounted and imaged by confocal microscopy (63× oil objective). Colocalization was analyzed with LAS X and ImageJ.

Bioluminescence imaging

C57BL/6 mice were intramuscularly injected in the hind limbs with DiR-labeled mRNA LNP, Free CpG mRNA LNP, and DSPE CpG mRNA LNP (3 µg of DiR per mouse), with a volume of 50 µL for each injection. An in vivo imaging system (Maestro EX, CRI Inc., Woburn, MA) was used to observe and analyze the biodistribution of different formulations at 0, 6, 12, 24, and 48 h.

Biodistribution of DSPE-CpG in vivo

C57BL/6 mice were intramuscularly injected in the hind limb with 5 µg per mouse of either Cy5-labeled DSPE-CpG or free Cy5-CpG (50 µL per injection). Six hours later, ex vivo fluorescence imaging of dissected tissues (including draining lymph nodes) was performed using an IVIS system to evaluate distribution, with quantification focused on lymph-node accumulation.

In vivo vaccination

For the SARS-CoV-2 model, male BALB/c mice (6–8 weeks old, n = 6 per group) were intramuscularly injected on day 0 with saline, mRNA LNP, Free CpG-mRNA LNP, or DSPE-CpG-mRNA LNP (10 µg RBD mRNA per mouse, 50 µL per hind leg). A booster was given on day 14. Blood was collected at indicated time points for serum isolation. Serum anti-RBD IgG was measured using a commercial ELISA kit (KIT006, Sino Biological). On day 28, spleens and inguinal lymph nodes were collected. Splenocytes (1 × 10⁷/mL) were restimulated with 5 µg/mL SARS-CoV-2 RBD protein for 48 h. Culture supernatants were analyzed for IFN-γ and IL-4 by ELISA. For intracellular cytokine staining, splenocytes were stained with fluorophore-conjugated antibodies against CD4, CD8, IFN-γ, IL-4, and IL-2 and analyzed by flow cytometry.

Pseudovirus neutralization assay

Neutralization was assessed using a SARS-CoV-2 spike pseudovirus (PSV001, Sino Biological) and 293T-ACE2 cells. Sera from immunized mice were heat-inactivated and serially diluted (starting 1:10). Diluted serum was mixed with an equal volume of pseudovirus, incubated (1 h, 37 °C), and added to 293T-ACE2 cells. After 48 h, cells were lysed, and luciferase activity was measured. Neutralization percentage was calculated relative to virus-only and cell-only controls. The half-maximal neutralization titer (NT₅₀) was determined using a four-parameter logistic model in GraphPad Prism 8.01.

Establishment of the hepatitis B model

Male C57BL/6 mice (6–8 weeks old) were injected via the tail vein with rAAV8-1.3HBV (1 × 10¹⁰ vg/mouse). Serum HBsAg was monitored weekly. Mice with HBsAg > 5000 IU/mL and HBeAg positivity at 4 weeks post-infection were selected for vaccination. These mice were randomly divided into four groups (n = 5) and immunized intramuscularly twice (days 0 and 7) with: saline, mRNA LNP (10 µg HBsAg mRNA), Free CpG-mRNA LNP, or DSPE-CpG-mRNA LNP.

Safety and stability profiles of DSPE CpG mRNA LNP

For acute toxicity, BALB/c mice were intramuscularly injected with DSPE-CpG-mRNA LNP (20 µg RBD mRNA). Serum ALT, AST, ALB, and CK levels were measured 24 h later. For histopathology, major organs were harvested on day 28, fixed, sectioned, and stained with H&E.

Statistical analysis

Statistical analyses were performed using GraphPad Prism 8.01 soft ware. The one-way analysis of variance (ANOVA) was used to assess the significance of differences among groups. The significance was defined as follows: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Result

Screening and optimization of the covalent linkage system of adjuvant - hydrophobic molecule

To evaluate the broad applicability of the hydrophobic modification strategy, conjugated the mouse-specific TLR9 agonist CpG 1826 and the humanized sequence CpG 1018 with octadecane (18 C) or 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) via amide bonds to construct amphiphilic adjuvants (Fig. 1 A). Cytotoxicity assays confirmed that neither modification exerted significant toxicity on DC2.4 cells, indicating their biocompatibility for subsequent studies. Flow cytometry analysis revealed the type of hydrophobic moiety modulated immune activation profiles. Activation of MHC class I costimulatory molecules was significantly stronger in the 18 C-CpG and free CpG groups than in the DSPE-CpG group (Fig. 1B). Conversely, the proportion of CD80⁺CD86⁺ double-positive dendritic cells (DCs) followed the gradient: 18 C-CpG > DSPE-CpG > free CpG (Fig. 1 C).

Fig. 1.

Construction and Screening of the Covalent Linkage System of Adjuvant-Hydrophobic Molecule. Schematic diagram of the covalent linkage system of adjuvant-hydrophobic molecule (A); Expression of MHC I molecules (B) and phenotypic maturation (C) of BMDCs stimulated by different concentrations of Free CpG, 18 C CpG and DSPE CpG, n = 3; Phenotypic maturation of BMDCs stimulated by different concentrations of Free CpG-mRNA LNP, 18 C CpG-mRNA LNP and DSPE CpG-mRNA LNP, n = 3 (D); Effects of different administration sequences of adjuvant and antigen on the phenotypic maturation of BMDCs, n = 3 (E); Loading efficiency of 18 C CpG and DSPE CpG on the surface of LNPs after co-incubation, n = 3 (F)

Critically, when formulated into mRNA-LNPs, DSPE-CpG conjugate induced significantly higher expression of maturation markers (CD80, CD86, MHC II) on BMDCs compared to free CpG (Fig. 1D), an effect corroborated by enhanced DC maturation in draining lymph nodes in vivo (Fig. 1E). This suggests that DSPE, as an inherent LNP component, improves adjuvant-antigen co-localization in both in vitro and in vivo settings. Therefore, DSPE-CpG was selected for further investigation.

We next quantified the loading efficiency of the conjugates onto pre-formed mRNA-LNPs. The loading efficiency of DSPE-CpG was 97.1% ± 2.1%, significantly higher than that of C18-CpG (64.2% ± 0.9%, p < 0.001; Fig. 1 F). This result provides direct evidence that the dual-chain phospholipid structure of DSPE enables more efficient and stable incorporation into the LNP bilayer compared to the single-chain C18 modifier.

Structural characterization and stability of DSPE - CpG - mRNA LNP

The DSPE-CpG-mRNA LNP system was successfully constructed via microfluidic technology (Fig. 2A). Incorporation of DSPE-CpG increased the particle size from 205.3 ± 3.7 nm to 222.7 ± 4.7 nm (Fig. 2B) and shifted the zeta potential to −11.37 ± 0.58 mV (p < 0.01), indicating phosphate headgroup exposure (Fig. 2D). Cryo-electron microscopy (cryo-EM) revealed that DSPE-CpG incorporation induced distinct lamellar nanostructures within the LNPs (Fig. 2E), likely due to its embedding into the lipid bilayer.

Fig. 2.

Flow chart of the application of the covalent linkage system of adjuvant-hydrophobic molecule in mRNA vaccines (A); particle size (B), polydispersity index (PDI) (C) and zeta potential (D) after the application of different covalent linkage systems of adjuvant-hydrophobic molecule in mRNA vaccines, n = 3; cryo-electron microscopy (cryo-EM) morphology showing lamellar nanostructures (E, scale bar = 200 nm) and agarose gel electrophoresis results (F) after the application of different covalent linkage systems of adjuvant-hydrophobic molecule in mRNA vaccines. RBD mRNA was used as the model nucleic acid; encapsulation efficiency of mRNA by LNPs after the addition of different adjuvants (G). Agarose gel electrophoresis analysis of DSPE CpG association with LNPs (H). Surface quenching efficiency of Cy5-labeled DSPE-CpG in different formulations determined by KI quenching assay, n = 3 (I)

Stability assessment showed that DSPE-CpG-LNPs resisted Triton X-100-induced mRNA degradation longer than formulations with free CpG (Fig. 2F). The mRNA encapsulation efficiency remained high (88.2%) after DSPE-CpG incorporation (Fig. 2G). Agarose gel electrophoresis confirmed the stable association of DSPE-CpG with LNPs, as the conjugate-LNP complex remained in the loading well, unlike free CpG or DSPE-CpG alone (Fig. 2H). Surface accessibility was further confirmed by KI quenching assay, where DSPE-CpG on LNPs exhibited significantly higher quenching efficiency (76.1%) than core-encapsulated DSPE-CpG (37.3%, p < 0.0001), demonstrating its surface-exposed localization (Fig. 2I).

The regulatory mechanism of DSPE - CpG on mRNA vaccine expression efficiency

Using an EGFP/RBD dual-reporter system, we assessed the effect of DSPE-CpG on mRNA translation. Fluorescence microscopy showed higher GFP intensity in 293 T, DC2.4, and BMDC cells treated with DSPE-CpG-EGFP mRNA LNP compared to the free CpG group (Fig. 3A). Flow cytometry analysis in 293 T cells revealed that the DSPE-CpG group maintained a high EGFP⁺ rate (86.2% ± 1.0%), comparable to the basic LNP group (90.7% ± 1.2%, p > 0.05), while the free CpG group showed a sharp decline (40.5% ± 4.1%, p < 0.001) (Fig. 3C). Notably, the proportion of high-expressing cells (fluorescence > 1 × 10⁵) was significantly greater in the DSPE-CpG group (15.8% ± 0.9%) than in the basic LNP (3.8% ± 1.2%) or free CpG (0.39% ± 0.1%) groups (Fig. 3E). In DC2.4 cells, while the total transfection rate was not significantly increased, the proportion of high-expressing cells was 1.94-fold higher with DSPE-CpG modification (67.5% ± 3.8%) than in the control (34.8% ± 3.7%) (Fig. 3F). Western blot confirmed that DSPE-CpG enhanced SARS-CoV-2 spike (RBD) protein expression by 3.25-fold compared to the basic LNP (Fig. 3B). This synergistic effect may stem from improved LNP membrane stability (encapsulation efficiency > 80%) and promoted clathrin-mediated endocytosis.

Fig. 3.

Evaluation of the in vitro transfection ability after the application of the covalent linkage system of adjuvant-hydrophobic molecule in mRNA vaccines. The expression of GFP protein in 293 T cells, DC2.4 cells and BMDCs cells at 24 h after transfection with mRNA LNP, Free CpG-mRNA LNP and DSPE CpG-mRNA LNP (A). The expression of eGFP protein in 293 T cells (C) and DC2.4 cells (D) was quantitatively analyzed by flow cytometry. When the fluorescence threshold was set to > 10⁵, the proportion of cells with high EGFP expression in 293 T cells (E) and the proportion of cells with high EGFP expression in DC2.4 cells (F). Effect of DSPE-CpG on the expression of SARS-CoV-2 spike protein (RBD) mRNA in 293 T cells (B)

Analysis of the intracellular transport mechanism

Using DiD fluorescent tracing, we monitored nanoparticle uptake in BMDCs. DSPE-CpG-mRNA LNP showed a pronounced uptake burst at 8 h, with fluorescence intensity 4-fold higher than that of the free CpG group (Fig. 4A). Inhibition assays confirmed the clathrin-dependence of this uptake; the proportion of clathrin-mediated endocytosis was 3.1-fold higher for the DSPE-CpG group than for the free CpG group (Fig. 4E). Lysosomal colocalization analysis indicated a trend towards enhanced endosomal escape for the DSPE-CpG group, although the Pearson correlation coefficient was not statistically different (Fig. 4C). These mechanisms explain the observed increases in high-expressing cells and protein levels.

Fig. 4.

Exploration of the intracellular mechanisms of DSPE CpG. Cellular uptake of different covalent linkage systems of adjuvant-hydrophobic molecules, n = 3, representative images (A), and analysis of image data by ImageJ (B); Confocal microscopy was used to observe the lysosomal escape of mRNA LNP, free CpG – mRNA LNP, and DSPE CpG – mRNA LNP in BMDCs at 24 h after administration. Cell nucleus (channel 1, blue); DiD-labeled nanoparticles (channel 2, red); Lysosomes (channel 3, pink); FAM-ssDNA (channel 4, green) (D). Scale bar: 25 μm. The images were analyzed by ImageJ, and the Pearson correlation coefficient between ssDNA and lysosomes was calculated, n = 3 (C); Cellular uptake of mRNA LNP, free CpG - mRNA LNP, and DSPE CpG - mRNA LNP after treating cells with three cellular uptake inhibitors, chlorpromazine, nystatin, and amiloride, n = 3 (E)

In vivo targeted distribution and immunomodulation

Biodistribution studies showed that DSPE-CpG-LNP achieved 1.68-fold and 1.36-fold higher fluorescence intensity in lymph nodes compared to the basic LNP and free CpG groups, respectively (p < 0.01; Fig. 5A-B). Using Cy5-labeled DSPE-CpG, we specifically traced the adjuvant and confirmed its superior lymph node targeting, with a 1.6-fold signal that of the free Cy5-CpG at 6 h post-injection (p < 0.05; Fig. 5C).

Fig. 5.

Distribution of DSPE-CpG in vivo. Representative biodistribution images (A) of mRNA vaccines with different covalent conjugation systems (adjuvant-hydrophobic molecules labeled as DiR) in BALB/c mice, and quantitative data in the liver, spleen, and lymph nodes, n = 5 (B); biodistribution and lymph-node targeting efficiency of Cy5-labeled CpG and DSPE-CpG in C57BL/6 mice, n = 5 (C)

Kinetics of the immune response and the advantage of Th1 polarization

Immunokinetic analysis revealed that DSPE-CpG-mRNA LNP increased DC density at the injection site by 1.29-fold at 6 h (Fig. 6A). By 12 h, it induced a T follicular helper (Tfh) cell activation rate of 7.1% ± 0.3%, and by 24 h, it achieved 2.38-fold higher DC maturity in lymph nodes than the basic LNP Fig. 6B.

Fig. 6.

Exploration of the in vivo mechanisms of DSPE CpG. Representative immunofluorescence staining images of the skin at the injection site 6 h after intramuscular administration of mRNA vaccines with different covalent linkage systems of adjuvant-hydrophobic molecules (A); The maturation of helper T cells in the bilateral inguinal lymph nodes 12 h after administration, n = 3 (B); The maturation of DCs in the bilateral inguinal lymph nodes 24 h after administration, n = 3 (C); The maturation of B cells in the bilateral inguinal lymph nodes 24 h after administration, n = 3 (D)

In the SARS-CoV-2 model, DSPE-CpG-LNP significantly enhanced Th1-type cellular immunity: the frequency of IFN-γ⁺CD8⁺ T cells (8.36% ± 1.8%) was 2.31-fold higher, and that of IL-2⁺CD4⁺ T cells (4.72% ± 0.8%) was 4.7-fold higher than in the basic LNP group (p < 0.001; Fig. 7C, F). Splenocyte restimulation showed 1.33-fold higher IFN-γ secretion in the DSPE-CpG group , confirming Th1 polarization (Fig. 7G-I).

Fig. 7.

Application of DSPE CpG complex in COVID-19 mRNA vaccine and induction of in vivo immune response. Schematic diagram of the experimental protocol (A); Flow cytometric analysis of the phenotypic maturation of DCs in the lymph nodes, n = 6 (B); Flow cytometric analysis of the frequencies of CD69⁺CD8⁺ T cells (C), IFN-γ⁺ CD4⁺ T cells (D), IL-4⁺ CD4⁺ T cells (E), and IL-2⁺ CD4⁺ T cells (F) after in vitro spike protein restimulation of SPLCs, n = 6. The levels of IFN-γ (G) and IL-4 (H) in the culture supernatant after in vitro spike protein restimulation of SPLCs were detected using an ELISA kit, and the ratio of the concentrations of IFN-γ to IL-4 (I) was calculated to analyze the phenotypic polarization of Th1 or Th2, n = 6; Flow cytometric analysis of IFN-γ⁺ CD4⁺ T cells (J), IL-4⁺ CD4⁺ T cells (K), and IL-2⁺ CD4⁺ T cells (L) after in vitro spike protein restimulation of SPLCs; The levels of anti-SARS-CoV-2 spike RBD antibodies in the serum of mice in each group were detected using an ELISA kit, n = 10 (M); Serum samples from mice in each group were collected, serially diluted, and the 50% pseudovirus neutralization titers (pVNT₅₀) were determined, n = 6 (N)

The DSPE-CpG-LNP formulation also induced a serum anti-RBD IgG concentration of 26.3 µg/mL, significantly higher than that induced by Free CpG-mRNA-LNP 19.4μg/mL or standard mRNA-LNP 18.1μg/mL (p < 0.01; Fig. 7M). Pseudovirus neutralization assays confirmed that all vaccine groups elicited significantly higher neutralization titers than the control, indicating enhanced protective potential (Fig. 7N).

Preliminary safety assessment

Safety profiling (Fig. 8A-D) showed no ALT/AST elevation between the DSPE-CpG-mRNA LNP group and the normal saline group (p > 0.05). After 28 days of chronic exposure, no pathological damage was observed in the main organs (Fig. 8E). Based on the above data, it strongly confirms that DSPE CpG - mRNA LNP has good safety.

Fig. 8.

Evaluation of the immunological safety after the application of different covalent linkage systems of adjuvant-hydrophobic molecules in mRNA vaccines. Peripheral serum was collected 28 days later for the detection of indexes including GGT (γ-glutamyl transpeptidase, A), TBIL (total bilirubin, B), alanine aminotransferase (ALT, C), CK (creatine kinase, D), ALB (albumin, E), and CR (serum creatinine, F), n = 6; Body weight was measured (G); Representative H&E staining images of the main organs of mice in different groups (H)

Verification of immunopotentiation effect using mRNA of hepatitis B surface antigen (HBsAg) as the model nucleic acid

To verify the broad-spectrum applicability of the hydrophobic modification strategy, this study further constructed a model of mRNA vaccine for hepatitis B surface antigen (HBsAg). Mice with chronic HBV infection were successfully established by tail vein injection of rAAV8-HBV (Fig. 9A). A chronic HBV replication model was successfully established. In HBV antigen models, DSPE-CpG-LNP induced 29.3 ± 2.1% CD107a⁺ CTLs, 9.1 ± 1.3% FasL⁺ CTLs and 12.4 ± 1.5% CD69⁺ CTLs (Fig. 9D-E), 2.10-, 1.8- and 1.5-fold higher than controls (p < 0.001). DC maturation reached 57.8 ± 4.3% (Fig. 9B), confirming cross-model consistency.

Fig. 9.

The application of the DSPE CpG complex in the hepatitis B virus mRNA vaccine and the induction of the in vivo immune response. Schematic diagram of the experiment (A); Flow cytometric analysis of the phenotypic maturation of DCs in the lymph nodes, n = 6 (B); Flow cytometric analysis of the frequencies of CD69⁺CD3⁺ T cells (C), CD107a⁺ CD8⁺ T cells (D), and FasL⁺ CD8⁺ T cells (E) after in vitro spike protein restimulation of SPLCs, n = 6

Discussion

This study establishes a covalent conjugation strategy that fundamentally addresses the co-delivery challenge in mRNA-LNP vaccines by stably tethering a TLR9 agonist (CpG) to a phospholipid (DSPE). The DSPE-CpG-mRNA-LNP platform demonstrates superior efficacy over conventional physical mixtures or simpler hydrophobic conjugates, primarily by ensuring the spatiotemporally coordinated delivery of antigen and adjuvant to the same antigen-presenting cells. This coordinated delivery, underpinned by enhanced nanoparticle stability and targeted biodistribution, unlocks robust and balanced cellular and humoral immunity.

The molecular design of the DSPE anchor is central to its functional advantage. Unlike the single alkyl chain of the C18 modifier, the dual C18 acyl chains of DSPE enable stable, transmembrane insertion into the LNP lipid bilayer [25, 26]. This critical structural distinction is corroborated by cryo-electron microscopy, which revealed an ordered lamellar nanostructure upon DSPE-CpG incorporation (Fig. 2E), and by a significant shift in zeta potential (Fig. 2D). This stable integration translates directly into quantitative benefits: the loading efficiency of DSPE-CpG onto LNPs was significantly higher than that of C18-CpG (97.1% vs. 64.2%, Fig. 2 F), and the formulation exhibited excellent physical and biological stability during long-term storage and freeze-thaw cycles (Supplementary Figs. S1-S6). Consequently, DSPE-CpG is presented on the LNP surface in a persistent and ordered manner, optimizing its availability for engagement with TLR9 on dendritic cells (DCs) and explaining its potent activation profile compared to the less stably anchored C18-CpG.

The covalently conjugated platform transforms the delivery paradigm from one of probable dissociation to guaranteed co-localization. Physically mixed free CpG, prone to dissociate from LNPs in vivo, results in suboptimal and asynchronous exposure of APCs to antigen and danger signals. This often leads to weak or skewed immune responses [27], as evidenced by the limited enhancement—and sometimes even inferior performance, of free CpG-mRNA-LNP over antigen-alone LNPs in stimulating certain T cell subsets (e.g., IL-2 + CD4+ T cells). In stark contrast, our DSPE-CpG-LNP system ensures that both components are delivered as an integral unit. This is mechanistically validated by its enhanced clathrin-mediated cellular uptake (Fig. 4E), superior lymph node targeting of the adjuvant itself (1.6-fold that of the free CpG, Fig. 5 C), and improved early antigen expression in vivo (Supplementary Fig. S11). The synchronized arrival of antigen and adjuvant within lymph node DCs creates an optimal immunogenic niche [28], driving robust and coordinated immune activation.

This mechanistic foundation is corroborated by consistently potent immune outcomes across diverse disease models. In the SARS-CoV-2 model, DSPE-CpG-LNP elicited a high-frequency, Th1-polarized response, characterized by a 2.31-fold increase in IFN-γ⁺ CD8⁺ T cells (Fig. 7) and significantly elevated levels of antigen-specific IgG with potent neutralizing activity (Fig. 7M, N). Similarly, in the therapeutic HBV model, it significantly amplified cytotoxic T lymphocyte (CTL) effector functions, marked by high frequencies of CD107a⁺ and FasL⁺CTLs (Fig. 9D,E). The convergence of these results across two distinct antigens underscores the versatility and reliability of our platform. The efficacy is traceable to its ability to induce robust DC maturation in lymphoid tissues (56.1% mature DCs in lymph nodes, Fig. 6 C), a prerequisite for efficient cross-presentation and the initiation of potent cellular immunity [29].

In summary, the DSPE-CpG-LNP platform is engineered on the principle of enforced co-delivery, which is achieved through covalent chemistry and rational lipid design. This strategy simultaneously solves two key problems in adjuvant delivery: it eliminates the dissociation bottleneck of physical mixtures and surpasses the integration instability of simpler hydrophobic modifications. By guaranteeing that the immunostimulatory signal (CpG) arrives together with the antigenic payload (mRNA) at the key site of immune induction, our platform ensures qualitatively superior APC programming. While future work may explore detailed intracellular trafficking kinetics and applications to other TLR agonists, this study robustly validates DSPE-CpG conjugation as a powerful and generalizable strategy, offering a compelling blueprint for next-generation mRNA vaccines requiring potent cellular immunity.

Author contributions

Wanting Ji: Conceptualization, Data curation, Formal analysis, Writing – original draft. Keer Zhu: Data curation, Writing – original draft. Yanyan Zhou: Conceptualization, Data curation, Investigation. Haomin Yan: Data curation, Formal analysis. Jiani Wei: Data curation, Formal analysis. Ying Zhu: Data curation, Formal analysis, Investigation. Sheng Li: Data curation, Formal analysis, Investigation. Yuqi Hong: Data curation, Formal analysis. Jinyao Dai: Data curation, Formal analysis. Shaohua Dong: Data curation, Formal analysis. Haoyang Hu: Data curation, Formal analysis. Jian You: Conceptualization, Writing – review & editing. Yunqing Qiu: Conceptualization, Writing – original draft, Writing – review & editing. Yan Lou: Conceptualization, Writing – original draft, Writing – review & editing.

Funding

This study was sponsored by Major Project under the Basic Public Welfare Research Program of Zhejiang Province (LD22H190003); International Cooperation and Exchange Programs of the National Natural Science Foundation of China (W2412017); Bill & Melinda Gates Foundation (2024VTJP1003); Key Project under the Zhejiang Provincial and Ministry Co-construction Plan (WKJ-ZJ-2425).

Data availability

Data will be made available on request.

Declarations

Ethics approval and consent to participate

All animal experiments in this study were strictly conducted in compliance with the Laboratory Animal Management Regulations of Zhejiang University and were approved by the Laboratory Animal Ethics Committee of Zhejiang University.

Consent for publication

We give our consent for the manuscript to be published in Journal of Nanobiotechnology.

Competing interests

The authors declare no competing interests.