Engineering of mRNA vaccine platform with reduced lipids and enhanced efficacy

Ma, Xu; Liu, Shaoli; Zhang, Shuhui; Liu, Zongran; Wang, Hui; Luo, Wendi; Zu, Mali; Qin, Hao; Li, Zhongxian; Zhong, Jie; Li, Junxi; Chen, Qizhe; Lin, Jiaqi; Liu, Andong; Zhang, Xinzheng; Li, Hongjun; Lu, Xueguang; Shi, Xinghua; Li, Lele; Gu, Zhen; Nie, Guangjun; Ji, Tianjiao

doi:10.1038/s41467-025-63965-3

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Published: 07 October 2025

Engineering of mRNA vaccine platform with reduced lipids and enhanced efficacy

Nature Communications volume 16, Article number: 8913 (2025) Cite this article

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Abstract

Lipid nanoparticles (LNPs) are the most clinically relevant vehicles for mRNA vaccines. Despite the great successes, the toxicity caused by the high dose of lipid components still represents a great challenge. The suboptimal loading capacity of mRNA in LNPs not only compromises the vaccine’s efficacy but also heightens the risk of non-specific immune responses, accelerates clearance caused by anti-PEG IgG/IgM. These problems underscore the urgent need for improving mRNA loading capacity in LNPs to provide dose-sparing effects. Herein, we develop a metal ion mediated mRNA enrichment strategy to efficiently form a high-density mRNA core, and manganese ion (Mn²⁺) exhibits a unique capability to match the need. The prepared Mn-mRNA nanoparticle is subsequently coated with lipids to form the resulting nanosystem, L@Mn-mRNA, which achieved nearly twice the mRNA loading capacity compared to conventional mRNA vaccine formulations (LNP-mRNA). Remarkably, L@Mn-mRNA also demonstrates a 2-fold increase in cellular uptake efficiency compared to LNP-mRNA, attributed to the enhanced stiffness provided by the Mn-mRNA core. By combining improved mRNA loading with superior cellular uptake, L@Mn-mRNA achieves significantly enhanced antigen-specific immune responses and therapeutic efficacy as vaccines. We elucidate the mechanism behind Mn-mRNA construction and optimize the L@Mn-mRNA formulations, and this method is suitable for types of lipids and mRNAs. Moreover, L@Mn-mRNA also reduces the risk of anti-PEG IgG/IgM generation. Thus, this strategy holds significant potential as a platform for the next generation of lipid-based mRNA vaccines.

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Introduction

In the past infectious disease pandemic, the mRNA vaccine stepped onto the stage of combating diseases^1,2,3,4. With the emergence of organ targeted mRNA delivery systems^5,6,7,8,9,10 and mRNA tumor vaccines^11,12,13, mRNA therapeutics are sprinting into a new era^14,15. Despite the great successes, the mRNA loading capacity in the vaccines still represents a great challenge. For example, the mRNA component was less than 4% in weight in COVID-19 BNT162b2 vaccine developed by Pfizer Inc. and BioNTech SE, and less than 5% in mRNA-1273 COVID-19 vaccine developed by Moderna, Inc.^16,17. In this case, to achieve the effective mRNA dose, the lipid dose will be relatively high. The toxicity and/or non-specific immune responses caused by the high dose of lipid components becomes a major concern of mRNA vaccines^18,19,20,21, such as high rate of headache (62.8% of individuals aged 18–64) and fever (17.4% of individuals aged 18–64), according to FDA data on mRNA-1273 COVID-19 vaccine²². Thus, besides of screening novel phospholipids^23,24,25,26 and optimizing the mRNA sequences^27,28, how to efficiently improve the mRNA loading capacity in LNP systems is also crucial and challenging for mRNA vaccines (and other mRNA therapeutics)^29,30.

Metal ions have been reported to assemble with short nucleic acids to form nanoparticles with high- nucleic acid loading content^31,32,33, which inspired us to apply this strategy in enriching mRNA in vaccines. As the proposed procedures shown in Fig. 1A, we envision that, if the condensed metal ions-mRNA nanoparticle (M-mRNA) is coated with lipids, the resulted nanoparticle (L@M-mRNA) would have a high-density mRNA core, which would achieve higher mRNA loading than the conventional LNP-mRNA complexes. However, the mRNA activity maintenance and the assembly efficiency are the crucial techniques.

**Fig. 1: Screening of metal ions-mediated mRNA enrichment.**

Herein, we developed a highly efficient mRNA enrichment strategy to improve the mRNA loading capacity in lipid-based vaccine. In brief, we explored several commonly used metal ions (Fe²⁺, Cu²⁺, Zn²⁺, and Mn²⁺) to prepare the M-mRNA complexes, and found that Mn²⁺ could enrich mRNA (termed Mn-mRNA nanoparticles) in high efficiency without influencing the mRNA activity. Mn-mRNA was subsequently coated with lipids, leading to nanosystems (L@Mn-mRNA) with an almost twofold mRNA loading capacity compared to conventional mRNA vaccine formulations (LNP-mRNA). Remarkably, L@Mn-mRNA achieved two times increase in cellular uptake efficiency compared to LNP-mRNA, attributed to the enhanced stiffness provided by the Mn-mRNA core. Therefore, L@Mn-mRNA achieved significantly improved antigen-specific immune responses and therapeutic efficacy as vaccines. We elucidated the mechanism of Mn-mRNA construction and optimization of the L@Mn-mRNA formulations to establish a potential platform for the lipid-based mRNA vaccine.

Results and discussion

Design and screening of high-mRNA-loading nanoparticles

To increase the mRNA loading, we tried to construct a metal ion-mRNA (M-mRNA) nanoparticle as the core to enrich mRNA before lipids coating (Fig. 1A). The reported nanoscale complexes made of metal ions and short nucleic acids were typically prepared at 95 °C for over 30 min^31,32,33. However, this harsh preparation condition may result in the degradation of mRNA. As shown in agarose gel electrophoresis results, EGFP mRNA (mEGFP), Luciferase mRNA (mLuc), and COVID-19 Spike protein mRNA (mSpike) degraded within 30 min at 95 °C (Fig. 1B and Supplementary Fig. 1), and the expression efficiency of mEGFP and mLuc in DC 2.4 cells (DCs) (transfected by Lipofectamine^TM 3000) was significantly reduced after heating (Fig. 1C). These results suggested that lower temperature and shorter incubation duration should be more favorable for enriching mRNA with metal ions. Therefore, we lowered the heating temperature to 65 °C, in which condition, the mRNA degradation and activity loss was significantly reduced, as demonstrated by the agarose gel (Fig. 1D and Supplementary Fig. 1) and DCs transfection experiments (Fig. 1E). Notably, over 95% of mRNAs (mEGFP, mLuc) maintained their activity within 5 min at 65 °C. Thus, we explored the potential for enriching mRNA under this condition.

Various metal ions were tested to assemble with mRNA at 65 °C. Among the Fe-mRNA, Cu-mRNA, Zn-mRNA, and Mn-mRNA complexes, only Mn²⁺ and Zn²⁺ groups could generate regular nanostructures (Fig. 1F). Then, we quantified the mRNA coordination ratio of Zn-mRNA and Mn-mRNA nanoparticles using a Quant-it^TM RiboGreen RNA Assay Kit^34,35. The results revealed that about 90% of mRNA formed nanoparticles in Mn-mRNA group within 5 min, whereas Zn-mRNA group required 15–30 min to reach similar efficiency (Fig. 1G). Considering the risk of mRNA degradation for prolonged treatment, we decided to use Mn²⁺ to enrich mRNAs at 65 °C for 5 min.

To investigate the mechanism of metal ions-mRNA complexes formation, we calculated the coordination bond lengths (l) and binding energy (ΔE) of metal ion with the adenine (A) and guanine (G) bases, which can in some extent reflect the stability of M-mRNA complex^36,37. The selected sites were where transition metal ions most readily bind with bases N7/O6 (for G) and N7 (for A)^36,37. As shown in Fig. 2A, Mn²⁺ and Zn²⁺ groups have longer bond lengths (no matter the l1, l2, and l3) than that of Fe²⁺ and Cu²⁺ groups, and Mn²⁺ have the lowest binding energy with bases compared with other metal ions. Shorter bond length and higher binding energy represents easier generation of stable M-mRNA complexes, which seems inconsistent with above results (that Fe²⁺ and Cu²⁺ groups did not form nanoparticles). We supposed that the formation of M-mRNA nanoparticles involved two steps: (1) binding, metal ions coordination with mRNA bases at room temperature, potentially forming complexes without regular morphologies; (2) rearrangement, the heating provided energy to break the metal ion-base coordination, leading to the reassembly of complexes and form nanoparticles (Fig. 2B). Thus, longer bond lengths (Mn²⁺ and Zn²⁺ with bases) and lower binding energy (Mn²⁺ with bases) indicated that lower temperature could realize the reassembly. To test our hypothesis, we monitored the heating process over 5 min and analyzed the spatial distribution of Mn element and P element (from mRNA) by using elemental analysis (EDS) mode of transmission electron microscopy (TEM). As shown in Fig. 2C, without heating, the reactants showed some aggregation (containing both Mn and P elements) and could not be collected by centrifugation, indicating that interactions between Mn and mRNA were already present. After 1 min of heating, no significant morphological change was observed, while particle morphology emerged at 3 min time point, though the structures was irregular. After 5 min heating, well-defined spherical nanoparticles were obtained. This observation supported our proposed mechanism of the M-mRNA formation (as shown in Fig. 2B), and the proposed coordination between the bases of mRNA and Mn²⁺ in the Mn-mRNA nanoparticles was shown in a 3D structure (Supplementary Fig. 2).

**Fig. 2: The mechanism of assembly from metal ions and mRNA.**

To investigate the relationship of Mn²⁺ and mRNA bases ratios in the Mn-mRNA formation, we tried to prepare the Mn-mRNA by using various molar ratios of these two components. Combining TEM images and dynamic light scattering (DLS) analysis, the system tended to generate uniform nanoparticles when the usage range of Mn²⁺ to mRNA base was controlled at 8:1 to 2:1 (Fig. 2D, Supplementary Fig. 3, and Supplementary Table 1), which was consistent with previous reports^31,32. Neither increasing nor decreasing Mn²⁺ ratio (e.g., Mn²⁺ to bases molar ratio of 20:1 or 1:1) was benefit for the nanoparticle formation. Insufficient Mn²⁺ probably cannot drive coordination with mRNA, while excessive Mn²⁺ may lead to aggregations among particles (Supplementary Fig. 4). Considering that the 5:1 ratio group had lower PDI, we used this input ratio in the following investigations. In addition, the integrity of mRNA in Mn-mRNA was verified by agarose gel electrophoresis analysis, which demonstrated no detectable mRNA degradation (fragmentation) in the Mn-mOVA lane (Supplementary Fig. 5, as red dashed rectangle shown).

To evaluate the availability of Mn²⁺ and mRNA during the preparation of Mn-mRNA nanoparticles, we quantitatively analyzed the percentage of input Mn²⁺ in Mn-mRNA nanoparticles by using inductively coupled plasma mass spectrometry (ICP-MS). The result showed that 5% of the input Mn²⁺ involved the formation of Mn-mRNA. Notably, over 88% of the input mRNA had been involved in the formation of Mn-mRNA detected by Quant-it^TM RiboGreen RNA Assay Kit, indicating a high efficiency in mRNA enrichment. Based on the above results, we calculated the weight percentage of Mn in Mn-mRNA nanoparticles was 4.4%, and the mRNA was 95.6% in weight (the detailed calculation process and results were presented on “Method” Section and Supplementary Table 2).

We respectively prepared Mn-mRNA with mSpike (4k nt), mLuc (1.9k nt), mOVA (1.6k nt), and mEGFP (1k nt) to verify the universality of the preparation conditions (65 °C heating for 5 min with 5:1 molar ratio of input Mn²⁺ to bases), though the particle size of Mn-mRNA had a decreasing trend with the mRNA length increasing. All groups could form regular nanoparticles (Fig. 2E and Supplementary Table 3), indicative of the universality of this approach.

Subsequently, we coated the Mn-mRNA with lipids (Fig. 3A) and explored the mass ratio of lipids (comprising four components) to the Mn-mRNA (mRNA mass instead of Mn-mRNA mass) ranging from 2.5:1 to 20:1. Upon lipids coating, the zeta potential of the particles increased and reached a positive value at a 10:1 ratio (15.5 ± 2.4 mV) (Fig. 3B). Further increasing the lipids ratio (e.g., 20:1) did not result in the further increase in zeta potential (13.1 ± 0.2 mV), while the polydispersity index (PDI) increased at ratios greater than 10:1 (Fig. 3B). In addition, the mRNA expression efficiency in DCs was enhanced with the increase of lipids usage till the ratio of lipids to Mn-mRNA reached 10:1 (Fig. 3C). Further increasing the lipids ratio did not significantly improve the transfection efficiency (relative mLuc expression: 1 ± 0.13 vs. 1 ± 0.09 for 10:1 vs. 20:1 of lipids to Mn-mRNA ratio). Therefore, we considered that the 10:1 ratio of lipids to Mn-mRNA could perform the sufficient coating and the mRNA encapsulation efficiency in this step was 98% (Supplementary Fig. 6). The mass ratio of Mn to mRNA in L@Mn-mRNA was consistent with that of Mn-mRNA nanoparticles (Supplementary Fig. 6). In which case, this formulation achieved a nearly 2-fold increase (9.1% vs. 4.7%) in mRNA loading capacity compared to current commercial products (Moderna mRNA-1273 COVID-19 vaccine) (Supplementary Table 4)³⁸.

**Fig. 3: Characterization of L@Mn-mRNA.**

Cryo-EM images showed a typical core-shell structure of lipid-coated Mn-mRNA (L@Mn-mRNA), indicating that Mn-mRNA nanoparticles were successfully encapsulated by lipids (Fig. 3D). Four types of mRNA (mSpike, mLuc, mOVA, and mEGFP) and two types of commercialized ionizable lipids (SM-102 and Dlin-MC3-DMA) were used to investigate the universality of our method. Both SM-102 and Dlin-MC3-DMA could encapsulate these four types of mRNA (Fig. 3D, Supplementary Fig. 7). DLS results indicated an increase in the hydrodynamic diameter of the nanoparticles upon lipids coating, and the charge shifted from negative to positive in all groups (Fig. 3E and Supplementary Tables 3 and 5). To further confirm compositions of the core-shell structure, we employed elemental analysis to study the element distribution in L@Mn-mRNA nanoparticles. Distinct boundary could be observed in the Mn distribution, while the range of P was significantly larger than that of Mn (Fig. 3F). This result further demonstrated that the Mn-mRNA core had been coated by lipids.

The size, PDI, and zeta potential of L@Mn-mRNA did not have significant changes when incubated with PBS for three weeks (Fig. 3G), indicating a good stability. Meanwhile, we also prepared LNP-mRNA according to the commercial formulation as the control group for subsequent experiments³⁸, and their morphology, size, zeta potential, and mRNA encapsulation efficiency were shown in Supplementary Fig. 8 and Supplementary Table 6. Besides, the cytotoxicity of L@Mn-mRNA and LNP-mRNA was evaluated on DCs (Fig. 3H). At the same mRNA dosage, the lipids content in L@Mn-mRNA is approximately half that of LNP-mRNA, in which test, the high concentration group (10 µg mRNA dose) of the L@Mn-mRNA had higher cell viability than that of LNP-mRNA, suggesting that L@Mn-mRNA with less lipids had the potential to reduce the cytotoxicity from formulation themselves when a higher therapeutic dosage was required. In another test, at equivalent lipids levels, L@Mn-mRNA showed no significant difference in cytotoxicity compared to LNP-mRNA at each concentration, indicating that the small amount of Mn²⁺ in L@Mn-mRNA did not induce cytotoxicity.

Enhanced cellular uptake of L@Mn-mRNA by dendritic cells

Then we investigated the cellular uptake of L@Mn-mRNA (first labeled the mRNA (mLuc) with Cy5). Under equivalent mRNA concentration (dose), the L@Mn-Cy5-mRNA group (half lipid content of the LNP-mRNA group) exhibited enhanced Cy5 uptake both at 4 (1.68 ± 0.10 fold) and 10 (1.80 ± 0.29 fold) hours (Fig. 4A and Supplementary Figs. 9A and 10A) imaged by using confocal laser scanning microscopy (CLSM). Flow cytometry results indicated that the Cy5 signal in L@Mn-Cy5-mRNA treated DCs was nearly twofold of that in LNP-Cy5-mRNA treated group (Fig. 4B, C and Supplementary Fig. 11A, B). In another experiment, we labeled DMG-PEG2K with Cy5 to prepare the LNP-mRNA and L@Mn-mRNA (named as Cy5-LNP-mRNA and Cy5-L@Mn-mRNA), and DCs were incubated with these two types of nanoparticles at the same mRNA dose (Cy5-labeling lipids in Cy5-L@Mn-mRNA was half dose of that in Cy5-LNP-mRNA) for 4 h and 10 h, respectively. Both CLSM and flow cytometry showed similar Cy5 signal intensity in each group (relative intensity at 4 h: 1 ± 0.16 vs. 0.98 ± 0.14; at 10 h: 1 ± 0.09 vs. 1.08 ± 0.05) (Fig. 4D–F and Supplementary Figs. 9B, 10B, and 11C, D). These results suggested that the cellular uptake efficiency of L@Mn-mRNA was about twofold of LNP-mRNA at the same mRNA dosage.

**Fig. 4: Cellular uptake of L@Mn-mRNA.**

We deduced that the enhanced cellular uptake was attributed to the enhanced stiffness provided by the Mn-mRNA core^39,40. Therefore, we used atomic force microscopy (AFM) in mechanical mode to measure the Young’s modulus of LNP-mRNA and L@Mn-mRNA (Fig. 4G). After randomly selecting twenty particles, we found that the Young’s modulus of L@Mn-mRNA was about 1.3 times higher than that of LNP-mRNA (Fig. 4H). This result supported our hypothesis that increased stiffness enhanced the cellular uptake.

Additionally, confocal microscopy results showed that the colocalization of Cy5 with lysosomes (lysotracker green) decreased after 10 h of incubation with the cells (Supplementary Fig. 9A, B), indicating that L@Mn-mRNA could achieve lysosomal escape, and the Pearson correlation coefficient analysis showed that L@Mn-mRNA had a superior efficiency of lysosomal escape than that of LNP-mRNA (Supplementary Fig. 12). This phenomenon is probably attribute to the increased stiffness of L@Mn-mRNA (compared to LNP-mRNA), which facilitated enhanced lysosomal escape efficiency⁴¹. Notably, since the Mn-mRNA core could dissociate under physiological conditions (at PBS or FBS) (Supplementary Fig. 13) due to the presence of abundant ions and proteins, the mRNA expression would not be affected after the lysosomal escape.

L@Mn-mRNA promoted maturation and antigen presentation of dendritic cells in vitro and in vivo

Before we investigated immune effect of L@Mn-mRNA vaccine platform, we used mLuc as the reporter mRNA to compare the in vitro and in vivo expression efficiency of L@Mn-mRNA and LNP-mRNA. We incubated these two types of nanoparticles with bone marrow-derived dendritic cells (BMDCs) for 12 and 24 h, and quantified bioluminescence intensity at each time point using a plate reader. The result showed that L@Mn-mLuc had nearly two times higher in vitro expression efficiency than LNP-mLuc (Supplementary Fig. 14). We subcutaneously injected L@Mn-mLuc and LNP-mLuc into the tail bases of C57BL/6 mice, followed by bioluminescence imaging using the in vivo imaging system (IVIS) at 6 and 24 h post-injection. The images indicated that L@Mn-mLuc group had 4.1 times higher expression efficiency in vivo than LNP-mLuc at 6 h and twofold of LNP-mLuc at 24 h (Fig. 5A and Supplementary Fig. 15), probably due to its superior capability entering cells. After 3-week storage, the mRNA expression efficiency in L@Mn-mLuc reduced to 75 %, while the LNP-mLuc group reduced to 69 % (Supplementary Fig. 16). This phenomenon further demonstrated a comparable stability between L@Mn-mLuc and LNP-mLuc. We also do the subcutaneous injections on both sides of the groin, and the bioluminescence signal (Supplementary Fig. 17A) had a similar trend with Supplementary Fig. 15. The tissues were collected post the 24 h imaging, and both groups exhibited heightened expression exclusively in the inguinal lymph nodes (iLN) proximal to the injection site (Supplementary Fig. 17B). Overall, due to the improved cellular uptake, the L@Mn-mRNA significantly enhanced the mRNA expression efficiency both in vitro and in vivo, validating its potential to be an efficient mRNA delivery platform. In addition, the incorporation of Mn²⁺ did not alter the stability of the L@Mn-mRNA.

**Fig. 5: Immune effects of L@Mn-mRNA by promoting maturation and antigen presentation of dendritic cells in vitro and in vivo.**

Then the capability of L@Mn-mOVA to activate the dendritic cells maturation and antigen presentation was evaluated in vitro and in vivo. BMDCs were respectively treated with PBS, LNP-mOVA, L@Mn-mOVA (half dosage) and L@Mn-mOVA. After 24-h incubation, the dendritic cells maturation markers, such as CD80, CD86, CD40, were detected by using flow cytometry (Fig. 5B–D), and the secretion of pro-inflammatory cytokines IL-6 and TNF-α in the cell culture medium was tested by ELISA kits (Fig. 5E, F). The results showed that the L@Mn-mOVA group was 1.2-fold expression of CD80, 1.2-fold expression of CD86, and 1.7-fold expression of CD40 compared to the LNP-mOVA (Fig. 5B–D). The IL-6 and TNF-α secretion was both approximately threefold of that in the LNP-mOVA group (Fig. 5E, F). The antigen presentation was characterized by measuring the frequency of OVA (SIINFEKL)-H2Kb complexes on the surface of BMDCs, and the L@Mn-mOVA group exhibited a nearly 1.7 times higher antigen presentation compared to LNP-mOVA group (Fig. 5G). Notably, even the L@Mn-mOVA (half dosage) group showed significant increases in these results compared to the LNP-mOVA group.

We also explored if the immune response enhancement caused by Mn²⁺, because Mn²⁺ can be act as an agonist of cGAS-STING pathway^42,43,44,45. However, we did not observe significant STING activation after incubating L@Mn-mOVA with RAW-Lucia ISG reporter cells (for STING activation detection^46,47) compared to the MnCl₂ treated groups (100 μM & 200 μM, as previous reported^48,49) (Supplementary Fig. 18), probably because the Mn²⁺ concentration in L@Mn-mOVA was much lower than that of MnCl₂ groups. For example, Mn²⁺ concentration was only 8.4 µM even the mRNA dose reached 10 µg/mL in L@Mn-mOVA formulation. In addition, no differences were detected between L@Mn-mOVA and LNP-mOVA groups in STING activation (Supplementary Fig. 18). Therefore, the enhanced immune response in the L@Mn-mOVA group was more likely attributed to high mRNA loading and improved cellular uptake.

To evaluate in vivo responses, each formulation (PBS, LNP-mOVA, L@Mn-mOVA with different dosages) was subcutaneously injected into the inguinal region of C57BL/6 mice. iLNs were harvested for flow cytometric analysis at the 24 h time point after administration. with the results showing that L@Mn-mOVA group exhibited 2.5-fold expression of CD80, 1.7-fold expression of CD86, and 4.1-fold expression of CD40 compared to the group treated with LNP-mOVA (Fig. 5H–J). As for the in vivo antigen presentation, the L@Mn-mOVA group achieved 1.9-fold SIINFEKL-H-2Kb-positive cells compared to the LNP-mOVA group (Fig. 5K). In addition, the level of these markers in the half dosage mOVA in L@Mn-mOVA group was comparable to LNP-mOVA group, and some makers (such as CD40⁺ and SIINFEKL-H-2Kb-positive cells) were higher than that of LNP-mOVA. The gating strategy of DCs maturation and antigen presentation are presented in Supplementary Fig. 19. To further verify whether Mn²⁺ exerts an adjuvant function in vivo, we subcutaneously administered formulations into the inguinal region of mice on Day 0, 3, and 6, and detected the expression of p-IRF-3 (cGAS-STING pathway’s downstream marker) in the iLNs on Day 13. No significant difference in p-IRF-3 expression was observed between the LNP-mOVA group and the L@Mn-mOVA group, indicating that the L@Mn-mOVA did not lead to additional activation of the STING-related pathway compared to LNP-mOVA treatment (Supplementary Fig. 20).

Therapeutic efficacy of L@Mn-mRNA vaccine in B16-OVA melanoma tumor model

To evaluate the anti-tumor efficacy of L@Mn-mRNA vaccine, the B16-OVA melanoma tumor model was established on C57BL/6 mice, and the mice were subcutaneously administered in the inguinal regions with PBS, LNP-mOVA (10 μg, representing 10 μg mOVA per mouse), L@Mn-mOVA (5 μg, representing half dosage 5 μg mOVA per mouse) and L@Mn-mOVA (10 μg, representing 10 μg mOVA per mouse) three times (on Day 4, 7, and 10) (Fig. 6A). The L@Mn-mOVA group exhibited higher efficiency on tumor-suppression than that of the LNP-mOVA group (Fig. 6B and Supplementary Fig. 21A), with improving median survival rate by 26% (46–58 days) (Supplementary Fig. 21B). The half dosage L@Mn-mOVA still exhibited a certain degree of superior anti-tumor effect compared to the LNP-mOVA group, extending median survival by 13% (46–52 days) (Fig. 6B and Supplementary Fig. 21B).

**Fig. 6: Enhanced anti-tumor efficacy from L@Mn-mRNA vaccine.**

Then we evaluated the antigen-specific immune response in blood and splenocytes. The L@Mn-mOVA (10 μg) group showed eightfold increase in the proportion of SIINFEKL tetramer⁺ cells within CD8⁺ T cells in the blood and 1.8-fold increase in the spleen compared to the LNP-mOVA group (Fig. 6C, D). Although the difference between the L@Mn-mOVA (5 μg) group and the LNP-mOVA group in the blood was negligible, the L@Mn-mOVA (5 μg) group showed a significant increase in SIINFEKL tetramer⁺ cells within CD8⁺ T cells compared to the LNP-mOVA group in the spleen (Fig. 6C, D). The corresponding gating strategy was presented in Supplementary Fig. 22. Further, splenocytes isolated from mice subjected to various treatment groups were analyzed through the IFN-ELISpot assay. The half-dose L@Mn-mOVA (5 μg) group and the L@Mn-mOVA group (10 μg) exhibited threefold and 3.5-fold increases in the number of spots compared to the LNP-mOVA group (Fig. 6E, F), respectively. There results demonstrated that the L@Mn-mOVA groups (including the half-dose group) effectively activated antigen-specific immune responses.

For the serum pro-inflammatory cytokines (IL-6, TNF-α, IFN-α, and IFN-β) detection, the L@Mn-mRNA group had a significant increase compared to the LNP-mOVA group (Fig. 6G–J), and the TNF-α and IFN-β in L@Mn-mOVA (5 μg) group were also higher than that of the LNP-mOVA group, indicating an enhanced immunogenic effectiveness.

Then the immune infiltration at the tumor site was assessed by flow cytometry. The L@Mn-mOVA (10 μg) group exhibited twofold ratio of CD3⁺CD8⁺ T cells and 1.8-fold ratio of CD3⁺CD4⁺ T cells compared to that of LNP-mOVA group (Fig. 6K, L). The L@Mn-mOVA (10 μg) group also exhibited 1.5-fold number of CD11c⁺ cells (antigen-presenting cells) (Fig. 6M). The L@Mn-mOVA (5 μg) group showed a significant increase in the number of CD3⁺CD8⁺ T cells, CD3⁺CD4⁺ T cells and similar number of CD11c⁺ cells compared to the LNP-mOVA group. Thus, compared to the LNP-mOVA group, the L@Mn-mOVA exhibited a more robust stimulation of the immune system in combating tumors. Additionally, the half-dose group, L@Mn-mOVA (5 μg) showed comparable levels of immune activation to LNP-mOVA group. The corresponding gating strategy was presented in Supplementary Fig. 23.

To evaluate the capability of L@Mn-mRNA on inducing long-term immune memory, we subcutaneously administered the formulations on Day 0, Day 3, and Day 6 to C57BL/6 mice without tumor inoculation, and the mice were sacrificed for analysis on Day 70 (Fig. 6N). At the same dosage, the L@Mn-mOVA group had a higher proportion of CD44^highCD62L^high T cells (central memory T cells) and CD44^highCD62L^low T cells (effector memory T cells) in the spleen compared to the LNP-mOVA group, indicating stronger immune memory (Fig. 6O, P). The gating strategy of central memory T cells and effector memory T cells was presented in Supplementary Fig. 24. Based on these results, the L@Mn-mOVA exhibited superior anti-tumor efficacy compared to LNP-mOVA at the same dosage. Even the L@Mn-mOVA (5 μg) group, which meant the lipid usage was a quarter of that in LNP-mOVA, achieved effective anti-tumor immune response.

Safety evaluation and bioeffects of L@Mn-mRNA vaccine

It is crucial to evaluate the safety of the L@Mn-mRNA vaccine platform. During the therapeutic period, no fluctuations were observed in the body weight of the mice (Supplementary Fig. 25), and we found no significant organic disease in the major organs (heart, liver, spleen, lung, and kidney) after treatment compared to that of healthy mice (Supplementary Fig. 26). In addition, blood biochemical criterion (alanine aminotransferase (ALT), aspartate aminotransferase (AST), total protein (TP), albumin (ALB), globulin (GLB), albumin/globulin ratio (A/G) and alkaline phosphatase (ALP) for evaluating liver functions; blood urea nitrogen (BUN) and creatinine (CR) for evaluating kidney function; triglycerides (TG) for cardiovascular health) did not had significant differences in all groups (Supplementary Fig. 27), which indicated the formulations did not affect the functions of major organs.

Manganese accumulation in organs is another safety concern of the L@Mn-mRNA vaccine platform, since high manganese accumulation in brain may cause neurotoxic⁵⁰. After subcutaneously administering L@Mn-mRNA at Day 0, 3, and 6, we measured the manganese content in major organs (heart, liver, spleen, lung, kidney, brain) and blood of mice on Day 13. Although there was a certain level increase of manganese content in the liver (1.18 ± 0.07 fold) and kidney (1.25 ± 0.17 fold) for the L@Mn-mRNA group compared to the control group (Supplementary Fig. 28), the manganese concentration remained within the normal range for mice (because manganese is a type of microelement)^51,52,53,54. For other organs, including the brain, no significant manganese accumulation was observed after the treatment of L@Mn-mOVA (Supplementary Fig. 28). These results demonstrated that the Mn²⁺ component in L@Mn-mOVA vaccine would not generate toxicity.

Meanwhile, we conducted antibody tests for anti-PEG on blood samples from each group of mice before and after treatment. The timeline of the anti-PEG IgM/IgG antibody titers measurement was presented in Fig. 7A. In the PBS group, anti-PEG IgM levels showed no significant change before and after administration. In the LNP-mRNA (10 μg) group, 60% of the mice exhibited at least a 20-fold increase in anti-PEG IgM levels. In comparison, 60% of the mice showed a tenfold increase in anti-PEG IgM level in L@Mn-mRNA (10 μg) group and only 40% of the mice had a fivefold increase in L@Mn-mRNA (5 μg) group. Similarly, as for the measurement of anti-PEG IgG, in the LNP-mRNA (10 μg) group, 80% of the mice exhibited a substantial increase (ranging from a minimum of sixfold to a maximum of ninefold). In the L@Mn-mRNA (10 μg) group, only 20% of the mice exhibited a notable elevation in antibody levels (ninefold), with the remaining mice displaying marginal increments. In the L@Mn-mRNA (5 μg) group, all mice had no significant increase (Fig. 7B). These results revealed that a reduced lipids input (in the L@Mn-mRNA formulation) would be benefit to avoid a higher production of anti-drug antibodies, which potentially offered an effective strategy to avoid the accelerated blood clearance.

**Fig. 7: Bioeffects of anti-PEG IgM/IgG in mice treated with each formulation.**

To further demonstrate that the L@Mn-mRNA vaccine platform could overcome the mRNA expression decrease caused by anti-PEG IgG/IgM mediated clearance, we subcutaneously injected LNP-mLuc and L@Mn-mLuc (10 μg mLuc per mouse) into the tail bases of C57BL/6 mice on Day 0, 3, and 6, and we measured mRNA expression efficiency on Day 13 and 20 (Fig. 7C, D). The L@Mn-mLuc group exhibited around 93% and 35 % mLuc expression (compared to the first injection) on Day 13 and Day 20, respectively. However, the LNP-mLuc group only had less than 15% mLuc expression (compared to the first injection) on Day 13 and Day 20. These results demonstrated that the L@Mn-mRNA could maintain the mRNA expression efficiency in a certain degree, which was probably because of the reduced risk of anti-PEG IgG/IgM generation.

In summary, we have developed a platform technique for improving mRNA loading in lipid-based nanoparticles. Mn²⁺ could efficiently enrich mRNA and form a high-density mRNA core before the lipid encapsulation. The developed platform (L@Mn-mRNA) integrated advantages on increased mRNA loading, enhanced cellular uptake and decreased the risk of lipid or PEG induced side-effects or rapid clearance. The platform technique could be suitable for types of novel ionizable lipids and mRNA. Future studies may focus on the scale-up preparation and the universality to diseases (for mRNA-based therapy). Thus, this work holds the potential to provide references for the next generation of lipid-based mRNA therapeutics.

Methods

Materials

All mRNAs were purchased from APE×BIO or TriLink Biotechnologies. Manganese (Ⅱ) chloride tetrahydrate was obtained from Alfa Aesar (China) Chemicals Co., Ltd. DMG-PEG2K (R-PEG-0021), Cy5-DMG-PEG2K (R-PF-0170), and DSPC (LP-R4-076) obtained from Xi’an Ruixi Biological Technology Co., Ltd. DLin-MC3-DMA was purchased from AVT (Shanghai) Pharmaceutical Tech Co., Ltd. Cholesterol was purchased from Solarbio Biotech Co., Ltd. SM-102 was purchased from Xiamen Sinopeg Biotech Co., Ltd. Hoechst 33342 (62249), LysoTracker® Green DND-26 (L7526) and Lipofectamine 3000 (L3000075) were purchased from Invitrogen Trading (Shanghai) Co., Ltd. FITC anti-mouse CD11c antibody (117305, 1:100), PE-Cy7 anti-mouse CD86 antibody (105013, 1:100), APC anti-mouse CD80 antibody (104713, 1:100), APC anti-mouse CD40 antibody (124611, 1:100), APC anti-mouse H-2Kb bound to SIINFEKL antibody (141605, 1:100), PE anti-mouse H-2Kb bound to SIINFEKL antibody (141603, 1:100), PE anti-mouse CD40 antibody (124609, 1:100), FITC anti-mouse CD3 antibody (100203, 1:100), PE-Cy7 anti-mouse CD8 antibody (100721, 1:100), APC anti-mouse CD4 antibody (100515, 1:100), APC anti-mouse CD11c antibody (117309, 1:100), APC anti-mouse CD44 antibody (103011, 1:100) and PE anti-mouse CD62L antibody (104407, 1:100) were purchased from Biolegend. T-Select H-2kb OVA Tetramer-SIINFEKL-PE mouse (TS-5001-1C) were purchased from MBL Life Science. IFN-1α (MM-47294M1) and IFN-1β (MM-46462M1) ELISA kit assay were obtained from Meimian Industrial Co., Ltd. Mouse Anti-PEG IgM ELISA (#PEGM-1) and Mouse Anti-PEG IgG ELISA (#PEGG-1) were purchased from Life Diagnostics, Inc. The mouse IL-6 (1210602), TNF-α (1217202), IFN-γ pre-coated ELISpot kit (2210005) were obtained from Dakewe Biotech Company. OVA257–264 (SIINFEKL) were obtained from Solarbio Biotech Co., Ltd. Phenylmethanesulfonyl fluoride was purchased from Beijing Lablead. D-Luciferin (D1007) was purchased from Solarbio Biotech Co., Ltd. Phospho-IRF-3 (Ser396) Rabbit mAb #29047 and Anti-rabbit IgG, HRP-linked Antibody 7074 (Goat) were purchased from Cell Signaling Technology. RPMI 1640 medium and Opti-MEM reduced serum medium were obtained from Thermo Fisher Scientific Inc.

Mn-mRNA and Lipid@Mn-mRNA (L@Mn-mRNA) preparation

For a reaction volume of 200 μL, we selected a 600 μL EP tube. First, 50 μL of 6 mM Mn²⁺ solution (manganese chloride tetrahydrate aqueous solution) was mixed with 130 μL of RNzyme-free water. Then, 20 μL of 1 mg/mL mRNA was combined with the former mixture. It was then vortexed for 6 min. Afterwards, the sample was heated in the metal bath at 65 °C for 5 min. After completion of the reaction, the sample was cooled to the room temperature. The Mn-mRNA nanoparticles were collected through centrifugation at 60,000 × g for 15 min, and the Mn-mRNA nanoparticles are dispersed in 200 μL of enzyme-free water. The mRNA and Mn²⁺ that were not included in Mn-mRNA nanoparticles could be removed though the centrifugation. SM-102 (or Dlin-MC3-DMA), cholesterol, DSPC, and DMG-PEG2000 were mixed according to the proportions specified in the formulation (molar ratio of 50:38.5:10:1.5) using anhydrous ethanol as the solvent. A volume ratio of anhydrous ethanol to aqueous phase (1:3) and a total lipid to mRNA mass ratio (10:1) was maintained. The aqueous phase was then rapidly injected into the ethanol phase with a pipette, followed by rapid pipetting for 150 times. It was allowed to stand at room temperature for 1 h. Finally, excess ethanol from the system was removed using a dialysis cup with a cut-off molecular weight of 10 kDa to obtain the L@Mn-mRNA.

For the preparation of Fe-mRNA, Cu-mRNA, and Zn-mRNA, 6 mM M²⁺ solutions were respectively prepared using ferric chloride tetrahydrate, anhydrous copper (II) sulfate, and zinc nitrate enneahydrate. The ratios of M²⁺ to bases were referred from the literature^31,32. The remaining steps were identical to the preparation method of Mn-mRNA. When preparing Mn-mRNA with different Mn²⁺ to nucleotide molar ratios, the amount of manganese ions was adjusted accordingly. When preparing L@Mn-mRNA with different total lipid-to-mRNA mass ratios, the amount of lipids was adjusted accordingly.

Mn-mRNA, L@Mn-mRNA, and LNP-mRNA characterization

The morphological characterization of the particles was imaged using transmission electron microscopy (Hitachi HT7700) and cryo-electron microscopy (Thermo Fisher Titan Krios), respectively. Distribution of phosphorus (green) and manganese (red) elements of particles was detected by Energy-Dispersive X-ray Spectroscopy (EDS) element analysis from field-emission transmission electron microscope (JEOL JEM-F200). The size, polydispersity index (PDI) and zeta potentials of Mn-mRNA, L@Mn-mRNA, and LNP-mRNA were measured by using DLS (Malvern Panalytical Mastersizer). Each set of data was tested three times. Diameters are reported as the number mean peak average. The characterization of particle size and zeta potential for all particles was conducted by diluting the particles in water at a ratio of 1:50. To calculate mRNA reaction efficiency, Zn-mRNA or Mn-mRNA nanoparticles and the corresponding supernatant were dissolved by mixing them with a hydrochloric acid solution at pH 3³⁴. The mRNA content in both particles and supernatant was measured separately using the Quant-it^TM RiboGreen RNA Assay Kit (n = 3 in each group). The Mn²⁺ content was measured using ICP-MS (Perkinelmer NexION 300X). The mRNA encapsulation efficiency of LNP-mRNA was calculated by Quant-it™ RiboGreen RNA Assay Kit according to the instructions.

The quantitative calculation of the Mn-mRNA composition was showed below:

1.
Mn ratio involved in the reaction (by ICP-MS) equals to “Mn in the precipitate (Mn-mRNA) (μg)”/“Mn in the precipitate (Mn-mRNA) (μg) + Mn in the supernatant solution (μg)”
2.
Mn amount involved in the reaction equals to “Mn Input × Mn ratio involved in the reaction”
3.
mRNA amount involved in the reaction equals to “mRNA Input” × “mRNA encapsulation efficiency”
4.
The proportion of mRNA in Mn-mRNA equals to “mRNA involved (μg)” / “Mn involved (μg) + mRNA involved (μg)”
5.
The proportion of Mn in Mn-mRNA equals to “Mn involved (μg)”/“Mn involved (μg) + mRNA involved (μg)”

L@Mn-mRNA was mixed with a solution containing 2% Triton ×-100 and hydrochloric acid (pH 3) to disrupt the lipid membrane and dissociate Mn-mRNA complexes. The mRNA content was then measured using the RiboGreen assay kit. The Mn content was determined by using ICP-MS, as previously described.

The mRNA capacity of L@Mn-mRNA was determined using the following formula:

mRNA capacity equals to “mRNA content/(Mn content + mRNA content + lipid content)”

The mRNA capacity of LNP-mRNA was determined using the following formula:

mRNA capacity equals to “mRNA content/(mRNA content + lipid content)”

Agarose gel electrophoresis

Agarose gel electrophoresis was run on a 1% agarose gel using 1× TAE as electrophoresis buffer at 100 V for 30 min. The mRNA sample amount in each well was 0.5 μg. Subsequently, we utilized ultraviolet light for imaging.

Simulated calculation

The geometric and electronic properties of metal ions-mRNA have been computed using the Gaussian 09 W program package at B3LYP/6-31 G(d,p) level⁵⁵. In order to ensure the feasibility of the calculation, we have adjusted the number of self-selected electrons in systems. After optimization, the length of bonds was measured by Mutifwn⁵⁶. Utilizing the iefpcm implicit solvent model, single-point energy calculations for the structures were performed employing the wb97xd⁵⁷ functional with the def2TZVP⁵⁸ basis set. Frequency calculations were carried out using the 6-311Gd⁵⁹ basis set to determine the binding energies of the complexes at 298 K for different components.

$${\Delta E}_{{bindin}g}={\Delta E}_{{complex}}-{\Delta E}_{{compound}1}-{\Delta E}_{{compound}2}$$

LNP-mRNA and L@Mn-mRNA Young’s moduli test from AFM

Initially, a circular mica sheet with a diameter of 1 cm was affixed securely onto an iron plate. Subsequently, 30 μL of LNP-mRNA and L@Mn-mRNA were incubated respectively on the mica sheet for 20 min. After gently washing the mica sheet with PBS, the sheet was then covered with 20 μL of PBS. AFM imaging and force measurements were carried out in aqueous medium at 26 ± 1 °C by using a Bruker Multimode-8 with NanoScope Analysis software (Version 1.9). The probe used for measurements was SCANASYST-FLUID + . AFM images were recorded with the following scanning parameters: scan rate, 0.977 Hz; peak force amplitude, 40 nm; peak force frequency, 2 KHz and spring constant, 0.7 N/m. The force between the tip and the sample was carefully maintained so as not to collapse the nanoparticles. The resolution was 256 × 256 pixels per AFM. Twenty data points were selected for quantitative analysis in each group.

Cells and animals

Female C57BL/6 (6–8 weeks old) were purchased from SPF Biotechnology. This study was approved by the ethics committee of the National Center for Nanoscience and Technology of China (approval number NCNST21-2103-0401). The animals were maintained in a controlled environment with a 12-h light/dark cycle. The ambient temperature was maintained at 22–24 °C, and the humidity level was kept at 40–60%. The B16-OVA cell line was purchased from Meisen Chinese Tissue Culture Collections (CTCC; cell line number CTCC-003-0259). The DC 2.4 cell line was obtained from the American Type Culture Collection (Manassas, VA, USA). The B16-OVA cells and DC 2.4 cells were cultured in RPMI 1640 containing 10% fetal bovine serum (FBS), Pen-Strep (100 U/mL and 100 μg/mL, respectively) solution at 37 °C with 5% CO₂. Raw-Lucia ISG cells (Raw-Lucia) were purchased from InvivoGen. BMDCs were obtained through the following steps. After euthanizing C57BL/6 mice (6–8 weeks), they were placed in 50 mL centrifuge tubes filled with 75% ethanol for 10 min for sterilization. Subsequently, the leg bones were removed, muscles were stripped, and bones on both sides were clipped. Bone marrow was then flushed into new culture dishes using a needle. Red blood cell lysis buffer was added afterwards, and the lysis was terminated after 3 min. Finally, cells were collected by centrifugation and resuspended in culture medium containing 20 ng/mL GM-CSF (Peprotech) and 20 ng/mL IL-4 (Peprotech), with changing medium every two days. On Day 6, the nonadherent cells were aspirated and incubated in 6-well plates with fresh medium for further investigation.

In vitro transfection

DC 2.4 cells were seeded in a 96-well plate at a density of 10⁴ cells per well. Different kinds of mRNAs were heated for different durations in a metal bath, then mixed with Lipofectamine™ 3000 Transfection Reagent according to the instructions to form complexes (n = 3 in each group). These complexes were then transfected into cells at a dose of 100 ng mRNA per well. After 24 h, the EGFP expression level was measured using flow cytometry (ACEA NovoExpress) with 10000 live cells in FITC channel. The luciferase mRNA expression level was detected by mixing an equal volume of cell suspension and the Bright-Lite Plus Luciferase Assay System (Vazyme), and then results were measured by a plate reader.

Cellular uptake and lysosomal escape

L@Mn-Cy5-Mrna (L@Mn-Cm) and LNP-Cy5-mRNA (LNP-Cm) were synthesized using Cy5-labeled mRNA. Cy5-L@Mn-mRNA (CL@Mn-m) and Cy5-LNP-mRNA (CLNP) were synthesized using Cy5-labeled DMG-PEG2K. The particles were separately incubated with DC 2.4 cells for 4 and 10 h. DC 2.4 cells were stained with Hoechst 33342 (1:500) for 5 min and washed with Opti-MEM medium three times. After that, DC 2.4 cells were stained with LysoTracker® Green DND-26 (1:10000) for 30 min and washed with Opti-MEM medium three times. The cellular uptake (Hoechst 33342/405 nm; Cy5/640 nm) and lysosome escape events (DND-26/488 nm) were captured using a CLSM (Olympus FV3000). The endosomal escape ratio was analyzed using an image analysis algorithm (ImageJ).

DC 2.4 cells were seeded in a 12-well plate at a density of 10⁵ cells per well. Subsequently, LNP-mRNA and L@Mn-mRNA nanoparticles were separately incubated with DC 2.4 cells at an equal mRNA dosage for varying durations (mRNA dose: 0.5 μg/mL). Finally, cell uptake was quantitatively analyzed using flow cytometry (ACEA NovoExpress) with 10,000 live cells in APC channel (n = 5 in each group).

In vitro and in vivo bioluminescence

L@Mn-mLuc and LNP-mLuc were respectively incubated in BMDCs for 12 and 24 h. Subsequently, an equal volume of cell suspension and the Bright-Lite Plus Luciferase Assay System (Vazyme) were mixed. Finally, the bioluminescence intensity was measured using a plate reader.

At 6 and 24 h after the injection of L@Mn-mLuc and LNP-mLuc (mRNA dose: 5 μg per mouse), mice were injected intraperitoneally with 0.2 mL d-luciferin (15 mg/mL in DPBS). Ten minutes later, the mice were imaged with an in vivo imaging system (PerkinElmer IVIS). Bioluminescence was quantified using the Living Image software (PerkinElmer).

Western blot analysis

GAPDH and p-IRF-3 protein expression in iLNs were analyzed by western blot. C57BL/6 mice were administered multiple subcutaneous injections of PBS, LNP-mOVA (10 μg), L@Mn-mOVA (5 μg), and L@Mn-mOVA (10 μg) into the inguinal region of mice on Day 0, 3, 6, and detected the expression of p-IRF-3 in the iLNs on Day 13. Specifically, on Day 13, the iLN of the mice were removed, homogenized, and proteins were extracted using lysis buffer. The lysate was then centrifuged in a pre-chilled centrifuge. Centrifugation was conducted at 4 °C and 11,000 × g for 20 min. After centrifugation, the supernatants were harvested for subsequent protein concentration measurement. Protein levels were determined using a BCA assay kit (Beyotime), following the protocol provided by the manufacturer. Equal amounts of protein (15 μg per sample) were separated via SDS-polyacrylamide gel electrophoresis and then transferred onto PVDF membranes. Membranes were blocked using 5% bovine serum albumin to prevent non-specific binding, and subsequently incubated with primary antibodies. After washing, membranes were treated with the appropriate secondary antibodies. Protein bands were detected using enhanced chemiluminescence reagents (Bio-Rad), and densitometric analysis of the bands was performed using ImageJ software. Each experimental group included three independent samples (n = 3).

Enzyme-linked immunosorbent assay (ELISA)

PBS, LNP-mOVA (2 μg/well), L@Mn-mOVA (1 μg/well), and L@Mn-mOVA (2 μg/well) were separately incubated with BMDCs in a 6-well plate (3 × 10⁵ cells per well) for 24 h. The supernatant was collected for the detection of IL-6 and TNF-α secretion from BMDCs. After diluting the supernatant fivefold, 50 µL of the test sample was added to the pre-coated plates. Subsequent steps were carried out according to the manufacturer’s instructions. Finally, the results were read using a plate reader.

For the measurement of cytokines in serum, blood samples were collected from each group of mice at the endpoint of treatment (Day 17). After preparing serum from the blood samples, the serum was diluted fivefold. Then 50 µL of the test sample was added to pre-coated plates. Subsequent steps were carried out as described above (n = 4 in each group).

L@Mn-mRNA promoted maturation and antigen presentation of DCs in vitro and in vivo

BMDCs were seeded into a 12-well plate (1 × 10⁵ cells per well) and then incubated with PBS, LNP-mOVA (mOVA dose: 1 μg/mL), L@Mn-mOVA (mOVA dose: 0.5 μg/mL), and L@Mn-mOVA (mOVA dose: 1 μg/mL) for 24 h. The medium from each group was collected for ELISA of TNF-α, IL-6 according to the manufacturer’s instructions. Meanwhile, cells were collected and stained with FITC anti-mouse CD11c antibody (1:100), PE-Cy7 anti-mouse CD86 antibody (1:100), APC anti-mouse CD80 antibody (1:100), APC anti-mouse CD40 antibody (1:100), and PE anti-mouse SIINFEKL-H-2kb antibody (1:100) for 30 min at 4 °C before the detection of flow cytometry (ACEA NovoExpress).

iLN from each mouse was harvested at 24 h post-injection of PBS, LNP-mOVA (10 μg mOVA per mouse), L@Mn-mOVA (5 μg mOVA per mouse), and L@Mn-mOVA (10 μg mOVA per mouse) at the inguinal regions and were gently mechanically disrupted using sterile pestles in RPMI 1640 medium in a 1.5 mL tube. The resulting cell suspensions were collected and stained with FITC anti-mouse CD11c antibody (1:100), PE-Cy7 anti-mouse CD86 antibody (1:100), APC anti-mouse CD80 antibody (1:100), PE anti-mouse CD40 antibody (1:100), and APC anti-mouse SIINFEKL-H-2kb antibody (1:100) for 30 min at 4 °C before being analyzed by flow cytometry (ACEA NovoExpress).

Anti-tumor effects in a subcutaneous B16-OVA cancer model

To investigate the anti-tumor efficacy of the L@Mn-mRNA vaccine in a murine melanoma model, C57BL/6 mice were subcutaneously injected with 1 × 10⁶ B16-OVA cells into the right flank on Day 0. Mice were randomly divided into four groups and subcutaneously administered with PBS, LNP-mOVA (10 μg mOVA per mouse), L@Mn-mOVA (5 μg mOVA per mouse), or L@Mn-mOVA (10 μg mOVA per mouse) at the bilateral inguinal regions on Days 4, 7, and 10. The PBS group served as a negative control. Tumor dimensions were measured every other day using vernier calipers, and tumor volumes were calculated as length × width² × 0.5. On Day 17, mice were sacrificed, and tumors were harvested, and digested into single-cell suspensions, and analyzed by flow cytometry to characterize immune cell infiltration. Cells were stained for 30 min at 4 °C with FITC anti-mouse CD3 (1:100), PE-Cy7 anti-mouse CD8a (1:100), APC anti-mouse CD4 (1:100), and APC anti-mouse CD11c (1:100), and then analyzed using a flow cytometer (ACEA NovoExpress).

Blood and spleens were collected to assess antigen-specific T cell responses. Single-cell suspensions were stained with T-Select H-2Kb OVA Tetramer-SIINFEKL-PE (1:100) and PE-Cy7 anti-mouse CD8a (1:100), followed by flow cytometric analysis (ACEA NovoExpress). Spleens were further processed for IFN-γ ELISpot assays. Briefly, splenocytes (1 × 10⁵ cells per well) were seeded into 96-well plates pre-coated with anti-mouse IFN-γ antibodies and stimulated with SIINFEKL peptide for 20 h. After incubation, plates were sequentially treated with a biotinylated detection antibody and streptavidin-conjugated ALP. Spots were developed using a substrate solution and quantified using an automated ELISpot reader (Dakewe Biotech).

To evaluate the biosafety of the L@Mn-mRNA vaccines, blood and major organs (heart, liver, spleen, lung, and kidney) were collected and subjected to H&E staining for examination.

Long-term immune memory test

The C57BL/6 mice (6–8 weeks, female) were subcutaneously administered with PBS, LNP-mOVA (10 μg mOVA per mouse), L@Mn-mOVA (5 μg mOVA per mouse), and L@Mn-mOVA (10 μg mOVA per mouse) at the right bilateral inguinal regions on Day 0, 3, and 6. To analyze memory T cells, splenocytes were collected at Day 70 (n = 5) and stained with FITC anti-mouse CD3 (1:100), PE-Cy7 anti-mouse CD8 (1:100), APC anti-mouse CD44 (1:100), and PE anti-mouse CD62L (1:100) antibodies. The central memory T cells (CD3⁺CD8⁺CD44^highCD62L^high) and effector memory T cells (CD3⁺CD8⁺CD44^highCD62L^low) were analyzed by flow cytometry (ACEA NovoExpress).

Anti-PEG IgG and IgM measurement

The C57BL/6 mice (6–8 weeks, female) were subcutaneously administered with PBS, LNP-mOVA (10 μg mOVA per mouse), L@Mn-mOVA (5 μg mOVA per mouse), and L@Mn-mOVA (10 μg mOVA per mouse) at the right bilateral inguinal regions on Day 0, 3, and 6. To detect Anti-peg IgM and Anti-peg IgG, blood of each group was respectively collected at Day −1, 13, and Day 20. After allowing the whole blood to stand and then centrifuging it to obtain plasma, the plasma was diluted 500-fold. ELISA assay kits are utilized to separately measure the antibody titers of Anti-peg IgM and Anti-peg IgG. Briefly, 100 μL diluted samples was dispensed into the coated wells. Then, 100 μL diluted HRP conjugate was added into each well. After dispensing 100 μL TMB reagent into each well, the plate was gently mix for 20 min. Finally, 100 μL stop solution was added, and the results were collected at a plate reader.

Statistical analysis

All results are presented as the mean ± S.D. from at least three independent experiments. Statistical differences between two groups were determined by two-tailed t-test, and the differences among three or more groups were determined by one-way ANOVA with the Tukey multiple comparison post-test. P values of P < 0.05 were regarded as significant. Animal survival rates were compared with the log-rank test using GraphPad Prism 9.0 (GraphPad Software, San Diego, CA, USA).

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

All data supporting the conclusions of this study are included in the manuscript and supplementary materials. Source data are provided with this paper. Source data is available for Figs. 1–7 and Supplementary Figs. 1–28 in the associated source data file Source data are provided with this paper.

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Acknowledgements

This work was supported by the National Key Research and Development Program of China (2021YFA0909900), the Beijing Natural Science Foundation (Z220022), and the National Natural Science Foundation of China (32271391). We sincerely acknowledge the valuable discussions with Ms. Yike Wu, Dr. Na Chen, Dr. Shuai Liu, Dr. Guangna Liu, and Dr. Keman Cheng. Elements of Figs. 1–4 and Supplementary Figs. 4 and 6 were created with BioRender.com and had got the publication license.

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CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, CAS Center for Excellence in Nanoscience, National Center for Nanoscience and Technology, Chinese Academy of Sciences, Beijing, China
Xu Ma, Shuhui Zhang, Zongran Liu, Mali Zu, Hao Qin, Zhongxian Li, Jie Zhong, Lele Li, Guangjun Nie & Tianjiao Ji
School of Nanoscience and Engineering, University of Chinese Academy of Sciences, Beijing, China
Xu Ma, Shuhui Zhang, Zhongxian Li, Jie Zhong, Lele Li, Guangjun Nie & Tianjiao Ji
Hangzhou Jitai Pharmaceutical Technology CO., Ltd, Hangzhou, China
Shaoli Liu & Andong Liu
Laboratory of Theoretical and Computational Nanoscience, National Center for Nanoscience and Technology, Chinese Academy of Sciences, Beijing, China
Hui Wang, Wendi Luo & Xinghua Shi
National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China
Junxi Li & Xinzheng Zhang
Beijing National Laboratory for Molecular Sciences, CAS Key Laboratory of Colloid, Interface and Chemical Thermodynamics, Institute of Chemistry, Chinese Academy of Sciences, Beijing, China
Qizhe Chen & Xueguang Lu
Key State Laboratory of Fine Chemicals, School of Bioengineering, Dalian University of Technology, Dalian, China
Jiaqi Lin
College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China
Hongjun Li & Zhen Gu

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Xu Ma
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Shuhui Zhang
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Contributions

X.M., T.J., and G.N. conceived the idea, analyzed the data, and wrote the manuscript. X.M., S.L., A.L., S.Z., Zongran Liu, M.Z., Zhongxian Li, J.Z., Junxi Li, Q.C., W.L. performed experiments. H.Q., Jiaqi Lin, and X.L. provided advice on in vivo experiments. X.S. and H.W. supervised and conducted the computational simulation portion. X.Z. and Junxi Li guided and implemented the cryo-electron microscopy section. All authors contributed to the discussion and editing of manuscript. Z.G., H.L., and L.L. provided suggestions for the overall work.

Corresponding authors

Correspondence to Lele Li, Zhen Gu, Guangjun Nie or Tianjiao Ji.

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X.M. and T.J. have filed the patent application on this research. The other authors declare no conflict of interest.

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Ma, X., Liu, S., Zhang, S. et al. Engineering of mRNA vaccine platform with reduced lipids and enhanced efficacy. Nat Commun 16, 8913 (2025). https://doi.org/10.1038/s41467-025-63965-3

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Received: 19 February 2025
Accepted: 02 September 2025
Published: 07 October 2025
Version of record: 07 October 2025
DOI: https://doi.org/10.1038/s41467-025-63965-3