Engineering of mRNA vaccine platform with reduced lipids and enhanced efficacy

Engineering of mRNA vaccine platform with reduced lipids and enhanced efficacy | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Engineering of mRNA vaccine platform with reduced lipids and enhanced efficacy Tianjiao Ji, Xu Ma, Shaoli Liu, Shuhui Zhang, Zongran Liu, Hui Wang, and 16 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4755456/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version 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 efficiency of mRNA in LNPs not only compromises the vaccine’s efficacy but also heightens the risk of non-specific immune responses, accelerates clearance from the bloodstream, and exacerbates side effects associated with the lipid carriers. These problems underscore the urgent need for improving mRNA loading in LNPs to provide dose-sparing effects. Herein, we developed a manganese ion (Mn²⁺) mediated mRNA enrichment strategy to efficiently form a high-density mRNA core, termed Mn-mRNA nanoparticle, which is subsequently coated with lipids. The resulting nanosystem, L@Mn-mRNA, achieved over twice the mRNA loading compared to conventional mRNA vaccine formulations (LNP-mRNA). Remarkably, L@Mn-mRNA also demonstrated 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 achieved significantly enhanced antigen-specific immune responses and therapeutic efficacy as vaccines. We elucidated the mechanism behind Mn-mRNA construction and optimized the L@Mn-mRNA formulations, and this method is suitable for types of lipids and mRNAs. Thus, this strategy holds significant potential as a platform for the next generation of lipid-based mRNA vaccines. Biological sciences/Biotechnology/Nanobiotechnology/Nanoparticles Physical sciences/Nanoscience and technology/Nanomedicine/Drug delivery manganese ions-mediated mRNA enrichment improved mRNA loading reduced lipids usage mRNA vaccine platform enhanced immune response Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 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 tumour vaccines 11 , 12 , 13 , mRNA therapeutics are sprinting into a new era 14 , 15 . Despite the great successes, the mRNA payload 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 . Recent study found that parts of LNPs are even empty (without mRNA loading) 18 , 19 . 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 20 , 21 , 22 , 23 , 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 vaccine 24 . Thus, besides of screening novel phospholipids 25 , 26 , 27 , 28 and optimizing the mRNA sequences 29 , 30 , how to efficiently improve the mRNA payload in LNP systems is also crucial and challenging for mRNA vaccines (and other mRNA therapeutics) 31 , 32 . Metal ions have been reported to assemble with short nucleic acids to form nanoparticles with high- nucleic acid loading content 33 , 34 , 35 , which inspired us to apply this strategy in enriching mRNA in vaccines. As the proposed procedures shown in Fig. 1 A, 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. Herein, we developed a highly efficient mRNA enrichment strategy to improve the mRNA loading in lipid-based vaccine. In brief, we explored several commonly used metal ions (Fe 2+ , Cu 2+ , Zn 2+ and Mn 2+ ) to prepare the M-mRNA complexes, and found that Mn 2+ could enrich mRNA (termed Mn-mRNA nanoparticles) in high efficiency without destroying the mRNA activity. Mn-mRNA was subsequently coated with lipids, leading to nanosystems (L@Mn-mRNA) with an over 2-fold 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. 1 A). The reported nanoscale complexes made of metal ions and short nucleic acids were typically prepared at 95°C for over 30 minutes 33 , 34 , 35 . 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 minutes at 95°C (Fig. 1 B, Figure S1 ), and the expression efficiency of mEGFP and mLuc in DC 2.4 cells (DCs) (transfected by Lipofectamine™ 3000) was significantly reduced after heating (Fig. 1 C, D). 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. 1 E, Figure S1 ) and DCs transfection experiments (Fig. 1 F, G). Notably, over 95% of mRNAs (mEGFP, mLuc) maintained their activity within 5 minutes 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 ( Figure S2 ). Then we do the centrifugation to collect the Mn-mRNA and Zn-mRNA nanoparticles to quantified the mRNA coordination ratio using a Quant-it™ RiboGreen RNA Assay Kit 36 , 37 . The results revealed that about 90% of mRNA formed nanoparticles in Mn-mRNA group within 5 minutes ( Table S1 ), whereas Zn-mRNA group required 15–30 minutes to reach similar efficiency ( Table S2 ). Considering the risk of mRNA degradation for prolonged treatment, we decided to use Mn 2+ to enrich mRNAs at 65°C for 5 minutes. 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 38 , 39 . The selected sites were where transition metal ions most readily bind with bases N7/O6 (for G) and N7 (for A) 38 , 39 . As shown in Fig. 1 H, Mn 2+ and Zn 2+ groups have longer bond lengths (no matter the l1 , l2 and l3 ) than that of Fe 2+ and Cu 2+ groups, and Mn 2+ 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 Fe 2+ and Cu 2+ groups did not form nanoparticles). We supposed that the formation of M-mRNA nanoparticles involved two steps: 1) metal ions coordination with mRNA bases at room temperature, potentially forming complexes without regular morphologies; 2) the heating provided energy to break the metal ion-base coordination, leading to the reassembly of complexes and form nanoparticles (Fig. 1 I). Thus, longer bond lengths (Mn 2+ and Zn 2+ with bases) and lower binding energy (Mn 2+ with bases) indicated that lower temperature could realize the reassembly. To verify our hypothesis, we analyzed the element distributions of Mn and phosphorus (P) element during assembly of Mn-mRNA by using elemental analysis (EDS) mode of transmission electron microscopy (TEM). As shown in Fig. 1 J and Figure S3 , Mn and P element had been co-localized in a disordered form before heating, and the disordered complexes reassembled into spherical nanoparticles after heating. This observation supported our proposed mechanism of the M-mRNA formation (as shown in Fig. 1 I). To investigate the relationship of Mn 2+ 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 Mn 2+ to mRNA base was controlled at 8:1 to 2:1 (Fig. 1 K, Figure S4 , Table S3 ), which was consistent with previous reports 33 , 34 . Neither increasing or decreasing Mn 2+ ratio (e.g. Mn 2+ to bases molar ratio of 20:1 or 1:1) was benefit for the nanoparticle formation. Considering that the 5:1 ratio group had lower PDI and around 100 nm in size (which are more conducive to cellular uptake) 40 , we prepared Mn-mRNA nanoparticles by using this input ratio in the following investigations. To evaluate the availability of Mn 2+ and mRNA during the preparation of Mn-mRNA nanoparticles, we quantitatively analyzed the percentage of input Mn 2+ in Mn-mRNA nanoparticles by using inductively coupled plasma mass spectrometry (ICP-MS). The result showed that 5% of the input Mn 2+ 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™ 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 Table S4 ). To verify the universality of the preparation conditions (65°C heating for 5 minutes with 5:1 molar ratio of input Mn 2+ to bases), we respectively prepared Mn-mRNA with mSpike (4k nt), mLuc (1.9k nt), mOVA (1.6k nt) and mEGFP (1k nt). All groups could form regular nanoparticles (Fig. 1 L, Table S5 ), indicative of the universality of this approach. Subsequently, we coated the Mn-mRNA with lipids (Fig. 2 A) 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. 2 B). 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. 2 B). 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. 2 C). 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, in which case, this formulation achieved a 2-fold increase (10:1 vs. 20:1) in mRNA loading compared to current commercial products (Moderna mRNA-1273 COVID-19 vaccine) ( Table S6 ) 19 . 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. 2 D). Four types of mRNA (mSpike, mLuc, mOVA and mEGFP) and two types of 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. 2 D, Figure S5 ). 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. 2 E, Table S5 , Table S7 ). 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. 2 F). 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 seven days (Fig. 2 G, Figure S6 ), indicating a good stability. Meanwhile, we also prepared LNP-mRNA according to the commercial formulation as the control group for subsequent experiments 19 , and their morphology, size, zeta potential, and mRNA encapsulation efficiency were shown in Figure S7 and Table S8 . Besides, the cytotoxicity of L@Mn-mRNA and LNP-mRNA was evaluated on DCs (Fig. 2 H). At the same mRNA dosage, the lipids content in L@Mn-mRNA is approximately half that of LNP-mRNA, in which test, the 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. 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 Mn 2+ 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 and 10 hours ( Fig. 3 A, Figure S8A ) 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 two-fold of that in LNP-Cy5-mRNA treated group (Fig. 3 B, C, Figure S9A, 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 hours and 10 hours, respectively. Both CLSM and flow cytometry showed similar Cy5 signal intensity in each group (Fig. 3 D-F, Figure S8B, Figure S9C, D ). These results suggested that the cellular uptake efficiency of L@Mn-mRNA was about 2-fold of LNP-mRNA at the same mRNA dosage. We deduced that the enhanced cellular uptake was attributed to the core-shell nanostructure, which resulted in an increased stiffness of nanoparticles 41 , 42 . Therefore, we used atomic force microscopy (AFM) in mechanical mode to measure the Young’s modulus of LNP-mRNA and L@Mn-mRNA (Fig. 3 G). 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. 3 H). 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 hours of incubation with the cells ( Figure S8A, 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 ( Figure S10 ). Notably, since the Mn-mRNA core could dissociate under physiological conditions (at PBS or FBS) ( Figure S11 ), 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 hours, 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 ( Figure S12 ). 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 (Fig. 4 A). The images indicated that L@Mn-mLuc group had 4.1 times higher expression efficiency in vivo than LNP-mLuc at 6 h and 2-fold of LNP-mLuc at 24 h ( Figure S13 ), probably due to its superior capability entering cells. We also do the subcutaneous injections on both sides of the groin, and the bioluminescence signal ( Figure S14A ) had a similar trend with Figure S13 . 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 ( Figure S14B ). 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. 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-hour incubation, the dendritic cells maturation markers, such as CD80, CD86, CD40, were detected by using flow cytometry (Fig. 4 B-D), and the secretion of pro-inflammatory cytokines IL-6 and TNF-α in the cell culture medium was tested by ELISA kits (Fig. 4 E and F ). The results showed that the L@Mn-mOVA group was 1.2-fold expression of CD80, 1.2-fold expression of CD86, and 1.5-fold expression of CD40 compared to the LNP-mOVA (Fig. 4 B-D). The IL-6 and TNF-α secretion was both approximately 3-fold of that in the LNP-mOVA group (Fig. 4 E, 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. 4 G). Notably, even the L@Mn-mOVA (half dosage) group showed significant increases in these results compared to the LNP-mOVA group. We also explored the potential immune response enhancement caused by Mn 2+ , because Mn 2+ can be act as an agonist of cGAS-STING pathway 43 , 44 , 45 , 46 . However, we did not observe significant increase of the STING protein expression in BMDCs ( Figure S15 ) after incubation with L@Mn-mOVA, which probably because that the small amount (0.4% in the whole formulation) Mn 2+ could not activate the cGAS-STING pathway. 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. Inguinal lymph nodes were harvested for flow cytometric analysis at the 24 h time point after administration. with the results showing that L@Mn-mOVA group exhibited 3.5-fold expression of CD80, 4.5-fold expression of CD86, and 4.1-fold expression of CD40 compared to the group treated with LNP-mOVA (Fig. 4 H-J). As for the in vivo antigen presentation, the L@Mn-mOVA group achieved 2.6-fold SIINFEKL-H-2Kb-positive cells compared to the LNP-mOVA group (Fig. 4 K). 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 BMDCs maturation and antigen presentation are presented in Figure S16 . Therapeutic efficacy of L@Mn-mRNA vaccine in B16-OVA melanoma tumour model To evaluate the anti-tumour efficacy of L@Mn-mRNA vaccine, the B16-OVA melanoma tumour 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. 5 A). The L@Mn-mOVA group exhibited higher efficiency on tumour-suppression than that of the LNP-mOVA group (Fig. 5 B, Figure S17A ), with improving median survival rate by 26% (46–58 days) ( Figure S17B ). The half dosage L@Mn-mOVA still exhibited a certain degree of superior anti-tumour effect compared to the LNP-mOVA group, extending median survival by 13% (46–52 days) (Fig. 5 B, Figure S17B ). Then we evaluated the antigen-specific immune response in blood and splenocytes. The L@Mn-mOVA (10 µg) group showed 8-fold 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. 5 C, 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. 5 C, D). Further, splenocytes isolated from mice subjected to various treatment groups were analyzed thourogh the IFN-ELISpot assay. The half-dose L@Mn-mOVA (5 µg) group and the L@Mn-mOVA group (10 µg) exhibited 3-fold and 3.5-fold increases in the number of spots compared to the LNP-mOVA group (Fig. 5 E, 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. 5 G-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 tumour site was assessed by flow cytometry. The L@Mn-mOVA (10 µg) group exhibited 2-fold ratio of CD3 + CD8 + T cells and 1.8-fold ratio of CD3 + CD4 + T cells compared to that of LNP-mOVA group (Fig. 5 K, L). The L@Mn-mOVA (10 µg) group also exhibited 1.5-fold number of CD11c + cells (antigen-presenting cells) (Fig. 5 M). 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 tumours. Additionally, the half-dose group, L@Mn-mOVA (5 µg) showed comparable levels of immune activation to LNP-mOVA group. 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 tumour inoculation, and the mice were sacrificed for analysis on Day 70 (Fig. 5 N). At the same dosage, the L@Mn-mOVA group had a higher proportion of CD44 high CD62L high T cells (central memory T cells) and CD44 high CD62L low T cells (effector memory T cells) in the spleen compared to the LNP-mOVA group, indicating stronger immune memory (Fig. 5 O, P). The gating strategy of central memory T cells and effector memory T cells was presented in Figure S18 . Based on these results, the L@Mn-mOVA exhibited superior anti-tumour 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-tumour immune response. Bioeffects and safety evaluation of L@Mn-mRNA vaccine It is crucial to evaluate the safety of our L@Mn-mRNA vaccine platform. During the therapeutic period, no fluctuations were observed in the body weight of the mice ( Figure S19 ), 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 ( Figure S20 ). 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 ( Figure S21 ), which indicated the formulations did not affect the functions of major organs. 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 and Anti-PEG IgG antibody titers measurement was presented in Fig. 6 A. 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 10-fold increase in anti-PEG IgM level in L@Mn-mRNA (10 µg) group and only 40% of the mice had a 5-fold 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 6-fold to a maximum of 9-fold). In the L@Mn-mRNA (10 µg) group, only 20% of the mice exhibited a notable elevation in antibody levels (9-fold), with the remaining mice displaying marginal increments. In the L@Mn-mRNA (5 µg) group, all mice had no significant increase (Fig. 6 B). 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 (ADA), which potentially offered an effective strategy to avoid the accelerated blood clearance. Conclusion In summary, we have developed a platform technique for improving mRNA loading in lipid-based nanoparticles. Mn 2+ could 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. The platform technique could be suitable for types of novel ionizable lipids and mRNA. Thus, this work holds the potential to provide references for the next generation of lipid-based mRNA vaccines. 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), 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 (PMSF) was purchased from Beijing Lablead. D-Luciferin (D1007) was purchased from Solarbio Biotech Co., Ltd. STING (D2P2F) Rabbit mAb (13647) and Anti-rabbit IgG, HRP-linked Antibody 7074 (Goat) were purchased from Cell Signaling Technology (CST). 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 Mn 2+ 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 minutes. Afterwards, the sample was heated in the metal bath at 65 °C for 5 minutes. After completion of the reaction, the sample was cooled to the room temperature. The Mn-mRNA nanoparticles were collected through centrifugation at 60000 g for 15 minutes, and the Mn-mRNA nanoparticles are dispersed in 200 μL of enzyme-free water. The mRNA and Mn 2+ 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 hour. 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 M 2+ solutions were respectively prepared using ferric chloride tetrahydrate, anhydrous copper (II) sulfate, and zinc nitrate enneahydrate. The ratios of M 2+ to bases were referred from the literature 33,34 . The remaining steps were identical to the preparation method of Mn-mRNA. When preparing Mn-mRNA with different Mn 2+ 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 dynamic light scattering (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 3 36 . The mRNA content in both particles and supernatant was measured separately using the Quant-it™ RiboGreen RNA Assay Kit (n = 3 in each group). The Mn 2+ 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. Three independent replicate experiments were performed. 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)” Agarose gel electrophoresis Agarose gel electrophoresis was run on a 1 % agarose gel using 1X TAE as electrophoresis buffer at 100 V for 30 minutes. 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 09W program package at B3LYP/6-31G(d,p) level 47 . 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 Mutifwn 48 . Utilizing the iefpcm implicit solvent model, single-point energy calculations for the structures were performed employing the wb97xd 49 functional with the def2TZVP 50 basis set. Frequency calculations were carried out using the 6-311Gd 51 basis set to determine the binding energies of the complexes at 298K for different components. 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 minutes. 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. Seven 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 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 (ATCC, 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 ℃ with 5% CO 2 . Bone Marrow-Derived Dendritic Cells ( 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 minutes 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 minutes. 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 10 4 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 hours, 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 hours. DC 2.4 cells were stained with Hoechst 33342 (1:500) for 5 minutes 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 minutes 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 10 5 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 10000 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 hours. 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 hours 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 β-actin and STING protein expression in BMDCs were analyzed by western blot. 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*10 5 cells per well) for 24 hours. Subsequently, cells were lysed on ice using cell lysis buffer containing protease and phosphatase inhibitors. The lysate was then centrifuged in a pre-chilled centrifuge. Centrifugation was conducted at 4 °C and 12,000 rpm for 20 minutes. After centrifugation, the supernatant was collected for protein quantification. The protein concentration of the samples was quantified using a bicinchoninic acid assay (BCA protein assay kit; Beyotime) according to the manufacturer’s instructions. Protein lysates (15 μg per sample) were resolved by SDS-PAGE and transferred to a polyvinylidene fluoride membrane. After being blocked in 5% BSA, the polyvinylidene fluoride membrane was incubated with primary antibodies followed by the specific secondary antibodies. Immunoreactive proteins were visualized using enhanced chemiluminescence reagents (Bio-Rad). Quantitively analysis of WB results were carried out using ImageJ software (n = 3 in each group). 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*10 5 cells per well) for 24 hours. The supernatant was collected for the detection of IL-6 and TNF-α secretion from BMDCs. After diluting the supernatant five-fold, 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 five-fold. 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 × 10 5 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 hours. The medium from each group was collected for ELISA of TNF-α, IL-6, IFN-1α and IFN-1β 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 minutes at 4 °C before the detection of flow cytometry (ACEA NovoExpress). iLN from each mouse was harvested at 24 hours 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), APC 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-tumour effects in a subcutaneous B16-OVA cancer model To evaluate the anti-tumour effect of the L@Mn-mRNA vaccine in a solid tumour model, 1 × 10 6 murine melanoma cells B16-OVA cells were injected subcutaneously into the right flank of C57BL/6 mice on Day 0. The mice 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 4, 7 and 10. Mice treated with PBS were used as a negative control group. The tumour volume was measured every other day using Vernier calipers and calculated by the following formula: tumour volume = length × 1/2 width 2 . The mice were euthanized on Day 17. The tumours were collected, and digested into single-cell suspensions to analyze the infiltrating immune cells by flow cytometry. The resulting cell suspensions were collected and stained with FITC anti-mouse CD3 antibody (1:100), PE-Cy7 anti-mouse CD8a antibody (1:100), APC anti-mouse CD4 antibody (1:100), PE anti-mouse F4/80 antibody (1:100) and APC anti-mouse SIINFEKL-H-2kb antibody (1:100) for 30 minutes at 4 °C before being analyzed by flow cytometry (ACEA NovoExpress). Blood and spleens were collected to analyze the proportion of specifical T cells that recognize the SIINFEKL antigen. Spleens were collected for IFN-γ ELISpot analysis. According to the manufacturer’s instructions, splenocytes were seeded in a 96 well plate (10 5 cells per well), pre-coated with a mouse anti-IFN-γ antibody and incubated with SIINFEKL peptide for 20 hours. A biotinylated antibody specific for IFN-γ and alkaline-phosphatase conjugated to streptavidin were subsequently used to detect the IFN-γ secreted by the re-stimulated T cells. By adding a substrate solution, visual spots were formed at the sites of captured IFN-γ, and automated spot quantification was caried out by Dakewe Biotech. Blood and the major organs, including heart, liver, spleen, lung and kidney, were collected for hematoxylin and eosin (H&E) staining to assess the safety of the L@Mn-mRNA vaccines. 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, PE-Cy7 anti-mouse CD8, APC anti-mouse CD44 and PE anti-mouse CD62L antibodies. The central memory T cells (CD3+CD8+CD44 high CD62L high ) and effector memory T cells (CD3+CD8+CD44 high CD62L 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 minutes. 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, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 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). Declarations Declaration of competing interest The authors declare no conflict of interest. Acknowledgments 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 Figure 1, 2 and 3 were created with BioRender.com and had got the publication license. Author 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., Z.L., M.Z., Z.L., J.Z., J.L., Q.C., W.L. performed experiments. H.Q., J.L. and X.L. provided advice on in vivo experiments. X.S. and H.W. supervised and conducted the computational simulation portion. X.Z. and J.L. guided and implemented the cryo-electron microscopy section. All authors contributed to the discussion and editing of manuscript. 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Technology","correspondingAuthor":false,"prefix":"","firstName":"Hui","middleName":"","lastName":"Wang","suffix":""},{"id":336795576,"identity":"3c4e0b17-7b1f-4aaa-8a0a-99e82ef6dce5","order_by":6,"name":"Wendi Luo","email":"","orcid":"","institution":"National Center for Nanoscience and Technology","correspondingAuthor":false,"prefix":"","firstName":"Wendi","middleName":"","lastName":"Luo","suffix":""},{"id":336795577,"identity":"cae00510-7c63-4323-8b4f-f5ad8ab04b9e","order_by":7,"name":"Mali Zu","email":"","orcid":"","institution":"National Center for Nanoscience and Technology","correspondingAuthor":false,"prefix":"","firstName":"Mali","middleName":"","lastName":"Zu","suffix":""},{"id":336795578,"identity":"2f4d0b8d-e1eb-4abd-ae90-be5530571cca","order_by":8,"name":"Hao Qin","email":"","orcid":"","institution":"National Center for Nanoscience and Technology, Beijing 100190, China","correspondingAuthor":false,"prefix":"","firstName":"Hao","middleName":"","lastName":"Qin","suffix":""},{"id":336795579,"identity":"f1a9c460-77f5-4da5-a75d-700be332a897","order_by":9,"name":"Zhongxian Li","email":"","orcid":"","institution":"National Center for Nanoscience and Technology","correspondingAuthor":false,"prefix":"","firstName":"Zhongxian","middleName":"","lastName":"Li","suffix":""},{"id":336795580,"identity":"503dba61-1fe6-4cc4-bf19-932426e52c64","order_by":10,"name":"Jie Zhong","email":"","orcid":"","institution":"National Center for Nanoscience and Technology","correspondingAuthor":false,"prefix":"","firstName":"Jie","middleName":"","lastName":"Zhong","suffix":""},{"id":336795581,"identity":"d3e63869-5c08-48b2-9a6f-cb0daf690b6d","order_by":11,"name":"Junxi Li","email":"","orcid":"","institution":"Institute of Biophysics, Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Junxi","middleName":"","lastName":"Li","suffix":""},{"id":336795582,"identity":"96bd3a54-cd25-4da4-aa4d-3befa11f8928","order_by":12,"name":"Qizhe Chen","email":"","orcid":"","institution":"Institute of Chemistry, Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Qizhe","middleName":"","lastName":"Chen","suffix":""},{"id":336795583,"identity":"2f4667e7-cb86-4f82-ac16-f161db47c773","order_by":13,"name":"Jiaqi Lin","email":"","orcid":"","institution":"School of Bioengineering, Dalian University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Jiaqi","middleName":"","lastName":"Lin","suffix":""},{"id":336795584,"identity":"c24321df-61b4-4e8e-a726-c9782a2f2878","order_by":14,"name":"Andong Liu","email":"","orcid":"","institution":"Hangzhou Jitai Pharmaceutical Technology CO., Ltd","correspondingAuthor":false,"prefix":"","firstName":"Andong","middleName":"","lastName":"Liu","suffix":""},{"id":336795585,"identity":"0e509098-1915-4d56-82e4-1f49229f2363","order_by":15,"name":"Xinzheng Zhang","email":"","orcid":"https://orcid.org/0000-0002-0114-0270","institution":"National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences.","correspondingAuthor":false,"prefix":"","firstName":"Xinzheng","middleName":"","lastName":"Zhang","suffix":""},{"id":336795586,"identity":"966b0c05-2406-4f97-9078-35f78de49c58","order_by":16,"name":"Hongjun Li","email":"","orcid":"https://orcid.org/0000-0002-1765-8445","institution":"Zhejiang University","correspondingAuthor":false,"prefix":"","firstName":"Hongjun","middleName":"","lastName":"Li","suffix":""},{"id":336795587,"identity":"16f851c5-839a-4a4c-84a9-a6318a36600d","order_by":17,"name":"Xueguang Lu","email":"","orcid":"","institution":"Institute of Chemistry, Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Xueguang","middleName":"","lastName":"Lu","suffix":""},{"id":336795588,"identity":"6c941618-27d8-4765-8080-f907b242d725","order_by":18,"name":"Xinghua Shi","email":"","orcid":"https://orcid.org/0000-0001-5012-3453","institution":"Laboratory of Theoretical and Computational Nanoscience, National Center for Nanoscience and Technology (NCNST)","correspondingAuthor":false,"prefix":"","firstName":"Xinghua","middleName":"","lastName":"Shi","suffix":""},{"id":336795589,"identity":"5cd20cfb-657d-4eba-93d6-683a521032fc","order_by":19,"name":"Lele Li","email":"","orcid":"https://orcid.org/0000-0001-8593-9292","institution":"National Center for Nanoscience and Technology","correspondingAuthor":false,"prefix":"","firstName":"Lele","middleName":"","lastName":"Li","suffix":""},{"id":336795590,"identity":"1a2b29e0-ade8-42b9-81c4-b587e1de736d","order_by":20,"name":"Zhen Gu","email":"","orcid":"https://orcid.org/0000-0003-2947-4456","institution":"Zhejiang University","correspondingAuthor":false,"prefix":"","firstName":"Zhen","middleName":"","lastName":"Gu","suffix":""},{"id":336795591,"identity":"090103ae-3e1b-4211-bf94-3b75bab4f5a8","order_by":21,"name":"Guangjun Nie","email":"","orcid":"https://orcid.org/0000-0001-5040-9793","institution":"National Center for Nanoscience and Technology, China","correspondingAuthor":false,"prefix":"","firstName":"Guangjun","middleName":"","lastName":"Nie","suffix":""}],"badges":[],"createdAt":"2024-07-17 10:25:35","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4755456/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4755456/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":61988867,"identity":"a3fa81eb-64cb-4a1b-b0fd-260bfb300218","added_by":"auto","created_at":"2024-08-08 02:16:50","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":949920,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScreening of metal ions-mediated mRNA enrichment. A.\u003c/strong\u003e Proposed procedures for increasing mRNA loading capacity through metal ion mediated mRNA enrichment. \u003cstrong\u003eB. \u003c/strong\u003eStability evaluation of EGFP and Luciferase mRNA at 95 ℃ analyzed by agarose gel electrophoresis. \u003cstrong\u003eC. \u003c/strong\u003eEGFP mRNA expression evaluation after treated at 95 ℃. Each group of mRNAs was transfected to DC 2.4 cells using Lipofectamine™ 3000 Transfection Reagent, and relative EGFP expression was analyzed by flow cytometry. Data were presented as mean ± SD, n = 3. \u003cstrong\u003eD.\u003c/strong\u003e Luciferase mRNA expression evaluation after being heated at 95 ℃. The transfect procedure was same with (B), and relative luciferase expression was analyzed by microplate reader after incubated with luciferin. Data were presented as mean ± SD, n = 3.\u003cstrong\u003e E.\u003c/strong\u003e Stability evaluation of EGFP and Luciferase mRNA at 65 ℃ for different duration. \u003cstrong\u003eF. \u003c/strong\u003eEGFP mRNA expression evaluation after being heated at 65 ℃. Procedures were same with (B). Data were presented as mean ± SD, n = 3. \u003cstrong\u003eG. \u003c/strong\u003emLuc expression evaluation after being heated at 65 ℃. Procedures were same with (B). Data were presented as mean ± SD, n = 3. \u003cstrong\u003eH. \u003c/strong\u003eThe coordination bond lengths (\u003cem\u003el\u003c/em\u003e) and binding energy (\u003cem\u003eΔE\u003c/em\u003e) between metal ions and adenine (A(N7)) or guanine (G(N7/O6)). \u003cstrong\u003eI. \u003c/strong\u003eSchematic illustration of the mechanism for Mn-mRNA nanoparticles formation at 65 ℃. The heating provided energy for the rearrangement of Mn\u003csup\u003e2+\u003c/sup\u003e and mRNA molecules. \u003cstrong\u003eJ.\u003c/strong\u003e The distribution of manganese and phosphorus elements of Mn-mRNA complexes before and after the heating detected by Energy-Dispersive X-ray Spectroscopy (EDS) element analysis. Scale bar in yellow, 500 nm; scale bar in white, 100 nm. \u003cstrong\u003eK. \u003c/strong\u003eMorphologies of Mn\u003csup\u003e2+\u003c/sup\u003e and mRNA complexes with different molar ratios of Mn\u003csup\u003e2+\u003c/sup\u003e to mRNA. Scale bar in 20:1 and 10:1 group, 500 nm, scale bar in other groups, 100 nm. \u003cstrong\u003eL. \u003c/strong\u003eMn-mRNA nanoparticles prepared at the same Mn\u003csup\u003e2+\u003c/sup\u003e to mRNA molar ratio (5:1) at 65 ℃ for 5 minutes with different length mRNAs. Scale bar, 100 nm. Statistical significance of \u003cstrong\u003eC, D, F \u003c/strong\u003eand\u003cstrong\u003e G\u003c/strong\u003e were analyzed by one-way ANOVA with Tukey’s correction. mLuc was used in \u003cstrong\u003eJ\u003c/strong\u003e and \u003cstrong\u003eK\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-4755456/v1/032956f5e07cd3e02d4f7c4a.png"},{"id":61989241,"identity":"ecbadd0e-ea80-405c-8bc4-6838d105301a","added_by":"auto","created_at":"2024-08-08 02:24:50","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":597407,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterization of L@Mn-mRNA. A.\u003c/strong\u003e Schematic illustration of L@Mn-mRNA preparation. \u003cstrong\u003eB.\u003c/strong\u003e Size, PDI and zeta potential of L@Mn-mRNA with different mass ratios of lipids to mRNA. Data were presented as mean ± SD, n = 3. \u003cstrong\u003eC.\u003c/strong\u003e Relatively bioluminescence intensity evaluation in DC 2.4 cells after incubation with different groups (different mass ratios of lipids to mRNA) of L@Mn-mRNA for 24 hours. Data were presented as mean ± SD, n = 4. \u003cem\u003eP\u003c/em\u003e = 0.9999. Statistical significance was analyzed by two-tailed t-test. \u003cstrong\u003eD. \u003c/strong\u003eCryo-EM images of L@Mn-mRNA. mSpike, mLuc, mOVA and mEGFP were respectively used for L@Mn-mRNA preparation. Scale bar, 50 nm. \u003cstrong\u003eE.\u003c/strong\u003e Size, PDI, and zeta potential of L@Mn-mRNA nanoparticles detected by DLS. Data were presented as mean ± SD, n = 3. \u003cstrong\u003eF.\u003c/strong\u003eDistribution of phosphorus (green) and manganese (red) elements of L@Mn-mRNA detected by Energy-Dispersive X-ray Spectroscopy (EDS) element analysis. There was no gap between two green (P) particles as shown by the single white dash line, and there was a gap between manganese regions labeled by two white dash lines. Scale bar, 10 nm. \u003cstrong\u003eG.\u003c/strong\u003e The stability evaluation of L@Mn-mRNA detected by DLS. The L@Mn-mRNA storage temperature was 4 ℃. The size and PDI had no significant changes in seven days. Data were presented as mean ± SD, n = 3. \u003cstrong\u003eH. \u003c/strong\u003eCytotoxicity evaluation of L@Mn-mRNA and LNP-mRNA. L@Mn-mRNA and LNP-mRNA were respectively incubated with DC 2.4 Cells at equal mRNA concentration (left) (0, 0.1, 1, 2, 5, 10 μg/mL) or total lipid concentration (right) (0, 2, 20, 40, 100, 200 μg/mL) for 24 h. The cytotoxicity was evaluated by using CCK-8 assay. Data were presented as mean ± SD, n = 4. Statistical significance was analyzed by two-tailed t-test. \u0026nbsp;mLuc was used in \u003cstrong\u003eB\u003c/strong\u003e, \u003cstrong\u003eC\u003c/strong\u003e, \u003cstrong\u003eF, G and H\u003c/strong\u003e. SM-102 was used as ionizable lipid in the formulations in \u003cstrong\u003eB-H\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-4755456/v1/5e9e911448a41d726a7cd32c.png"},{"id":61988869,"identity":"2aa95b67-7c85-4bde-abaf-4a7e64662487","added_by":"auto","created_at":"2024-08-08 02:16:50","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":808977,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCellular uptake of L@Mn-mRNA. A.\u003c/strong\u003e Cellular uptake of LNP-Cy5-mRNA and L@Mn-Cy5-mRNA at 4 hours imaged by CLSM. Same Cy5-mRNA dose (0.5 μg/mL) was used to incubate with DC 2.4 cells. Red, Cy5 labeled mRNA; green, Lysotracker green; blue, Hoechst. Scale bar, 10 µm. \u003cstrong\u003eB.\u003c/strong\u003e Cellular uptake of LNP-Cy5-mRNA and L@Mn-Cy5-mRNA at 4 h time point detected by flow cytometry. \u003cstrong\u003eC. \u003c/strong\u003eQuantitative analysis of cellular uptake from flow cytometry. Mean fluorescence intensity of L@Mn-Cy5-mRNA was two-fold of that in LNP-Cy5-mRNA. Data were presented as mean ± SD, n = 5. \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001. \u003cstrong\u003eD. \u003c/strong\u003eCellular uptake of Cy5-LNP-mRNA and Cy5-L@Mn-mRNA at 4 h time point imaged by CLSM. Cy5 labeled lipid (Cy5-DMG-PEG2K) was used in this experiment. Same mRNA dose (0.5 μg/mL) was used, which meant the Cy5-DMG-PEG2K in Cy5-L@Mn-mRNA was about 50% of that in Cy5-LNP-mRNA. Red, Cy5 labeled DMG-PEG2K; green, Lysotracker green; blue, Hoechst. Scale bar, 10 µm. \u003cstrong\u003eE.\u003c/strong\u003e Cellular uptake of Cy5-LNP-mRNA and Cy5-L@Mn-mRNA at 4 h time point detected by flow cytometry. \u003cstrong\u003eF.\u003c/strong\u003e Quantitative analysis of cellular uptake from flow cytometry. Mean fluorescence intensity of Cy5-L@Mn-mRNA was similar to that of Cy5-LNP-mRNA. Data were presented as mean ± SD, n = 5. \u003cstrong\u003eG, H. \u003c/strong\u003eStiffness of L@Mn-mRNA and LNP-mRNA measured by AFM. Scale bar in AFM images, 200 nm. The Young’s modulus of L@Mn-mRNA was 1.3-fold of that in LNP-mRNA. Twenty nanoparticles of each group were randomly collected to calculate the stiffness. Data were presented as mean ± SD, n = 20. \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.0001. SM-102 and mLuc were used. Statistical significance of \u003cstrong\u003eC\u003c/strong\u003e, \u003cstrong\u003eF \u003c/strong\u003eand \u003cstrong\u003eH\u003c/strong\u003e was analyzed by two-tailed t-test.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-4755456/v1/5435a129babff81a20fb2a9d.png"},{"id":61988864,"identity":"95023c92-ea83-43e2-a73f-99ca40097117","added_by":"auto","created_at":"2024-08-08 02:16:50","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":245688,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImmune effects of L@Mn-mRNA by promoting maturation and antigen presentation of dendritic cells \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eand \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. A.\u003c/strong\u003e \u003cem\u003eIn vivo\u003c/em\u003e Luciferase expression of LNP-mLuc and L@Mn-mLuc at 6 hours after subcutaneous injection to mice imaged by IVIS (5 μg mLuc per mouse). The bioluminescence signal in L@Mn-mLuc group was about 4.1-fold stronger than that of LNP-mLuc group. Data were presented as mean ± SD, n = 3. \u003cem\u003eP\u003c/em\u003e = 0.0282.\u003cstrong\u003e \u003c/strong\u003eStatistical significance was analyzed by two-tailed t-test. \u003cstrong\u003eB-D.\u003c/strong\u003eFlow cytometry examination of BMDCs maturation percentage (CD80+, CD86+, CD40+ cells percentages) 24 hours after incubation 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). Data were presented as mean ± SD, n = 4. \u003cstrong\u003eE, F.\u003c/strong\u003e IL-6 (E) and TNF-α (F) secretion from BMDCs after treatment with each formulation for 24 hours detected by ELISA Kit. Data were presented as mean ± SD, n = 4. \u003cstrong\u003eG.\u003c/strong\u003e Flow cytometry analysis of SIINFEKL-H2Kb+ presentation on BMDCs after treatment with each formulation for 24 hours. Data were presented as mean ± SD, n = 4. \u003cstrong\u003eH-J.\u003c/strong\u003eFlow cytometry analysis of matured dendritic cells (CD86+, CD80+, CD40+) in LNs after administration with PBS, LNP-mOVA (mOVA dose: 10 μg per mouse), L@Mn-mOVA (half dosage, mOVA dose: 5 μg per mouse) and L@Mn-mOVA (mOVA dose: 10 μg per mouse) for 24 hours. Data were presented as mean ± SD, n = 4. \u003cstrong\u003eK.\u003c/strong\u003e Flow cytometry analysis of SIINFEKL-H2Kb+ presentation on matured dendritic cells in lymph nodes with each formulation for 24 hours. mOVA dose was the same with that of \u003cstrong\u003e(H-K\u003c/strong\u003e). Data were presented as mean ± SD, n = 4. SM-102 was used in this part. Statistical significance of \u003cstrong\u003eB-K\u003c/strong\u003e was analyzed by one-way ANOVA with Tukey’s correction.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-4755456/v1/93af6bcbb95251b284bdb9a4.png"},{"id":61989240,"identity":"510c30f5-5c45-4156-883d-249df32dfeb0","added_by":"auto","created_at":"2024-08-08 02:24:50","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":565965,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEnhanced anti-tumour efficacy from L@Mn-mRNA vaccine. A.\u003c/strong\u003e Time line of therapeutic procedures. \u003cstrong\u003eB.\u003c/strong\u003e Tumour growth curves of each mouse after treatment with each formulation. mOVA dose in LNP-mOVA and L@Mn-mOVA were 10 μg per mouse, mOVA dose in L@Mn-mOVA half dose was 5 μg per mouse. n = 5 in each group. \u003cstrong\u003eC. \u003c/strong\u003eSIINFEKL tetramer+ in CD8+ cells in the whole blood after the treatment (at Day 17) analyzed by using flow cytometry. Data were presented as mean ± SD, n = 5. \u003cstrong\u003eD. \u003c/strong\u003eFlow cytometry analysis of SIINFEKL tetramer+ in CD8+ cells in the spleen after the treatment (at Day 20). Data were presented as mean ± SD, n = 5. \u003cstrong\u003eE, F.\u003c/strong\u003e The IFN-γ secretion by splenocytes after restimulation with OVA peptide was determined by the ELISpot assay. Data were presented as mean ± SD, n = 5. \u003cstrong\u003eG-J.\u003c/strong\u003e Serum levels of IL-6 (G), TNF-α (H), IFN-β (I), and IFN-α (J) were evaluated by using ELISA kits. Data were presented as mean ± SD, n = 4. \u003cstrong\u003eK-M.\u003c/strong\u003e The infiltrating immune cells in tumours of each group after treatment (at Day 17) detected by using flow cytometry, including CD3+CD4+ T cells (K), CD3+CD8+ T cells (L), and CD11c+ DCs (M). Data were presented as mean ± SD, n = 4. \u003cstrong\u003eN. \u003c/strong\u003eTime line of immune memory measurement.\u003cstrong\u003e O, P.\u003c/strong\u003e Immune memory evaluation on Day 70 after treatment by flow cytometry. The proportions of central memory T cells (CD3+, CD8+, CD44\u003csup\u003ehigh\u003c/sup\u003e and CD62L\u003csup\u003ehigh\u003c/sup\u003e) (O) and effector memory T cells (CD3+, CD8+, CD44\u003csup\u003ehigh\u003c/sup\u003e, CD62L\u003csup\u003elow\u003c/sup\u003e) (P) in the splenocytes detected by using flow cytometry. Data were presented as mean ± SD, n = 5. SM-102 was used in this part. Statistical significance of \u003cstrong\u003eC\u003c/strong\u003e, \u003cstrong\u003eD\u003c/strong\u003e, \u003cstrong\u003eF\u003c/strong\u003e-\u003cstrong\u003eM\u003c/strong\u003e and \u003cstrong\u003eO\u003c/strong\u003e-\u003cstrong\u003eP \u003c/strong\u003ewere analyzed by one-way ANOVA with Tukey’s correction.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-4755456/v1/4f6ed5316a6b2ff134f66803.png"},{"id":61988865,"identity":"cce8b60c-2602-4c53-a888-ce9dcbf0bdb1","added_by":"auto","created_at":"2024-08-08 02:16:50","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":134105,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnti-PEG IgM and IgG level from each formulation treated mice. A.\u003c/strong\u003e The timeline of the Anti-PEG IgM and Anti-PEG IgG titers.\u003cstrong\u003e B. \u003c/strong\u003eAnti-PEG IgM and IgG measurement detected by ELISA Kit. Anti-PEG IgM was detected on Day -1 and Day 13, and Anti-PEG IgG was detected on Day -1 and Day 20. Day -1 represented the day before performing the injection of each formulation. SM-102 was used in this experiment. Data were presented as mean ± SD, n = 5.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-4755456/v1/ae852c054a4e84f8fe9dc4c9.png"},{"id":66676113,"identity":"0c37e00b-5e44-4a31-a510-358e6c4b0013","added_by":"auto","created_at":"2024-10-15 11:13:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4188151,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4755456/v1/8c8b0788-8784-495b-89a8-a13ed06add4c.pdf"},{"id":61988870,"identity":"193ddb39-f939-457e-9517-9a881925d074","added_by":"auto","created_at":"2024-08-08 02:16:50","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":6503622,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"SImRNAenrichmentNatNano20240717TJsubmission.docx","url":"https://assets-eu.researchsquare.com/files/rs-4755456/v1/5ae76940db18b3c858fea944.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Engineering of mRNA vaccine platform with reduced lipids and enhanced efficacy","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn the past infectious disease pandemic, the mRNA vaccine stepped onto the stage of combating diseases\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. With the emergence of organ targeted mRNA delivery systems\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e and mRNA tumour vaccines\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, mRNA therapeutics are sprinting into a new era\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Despite the great successes, the mRNA payload 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.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Recent study found that parts of LNPs are even empty (without mRNA loading)\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. 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\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, such as high rate of headache (62.8% of individuals aged 18\u0026ndash;64) and fever (17.4% of individuals aged 18\u0026ndash;64), according to FDA data on mRNA-1273 COVID-19 vaccine\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Thus, besides of screening novel phospholipids\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e and optimizing the mRNA sequences\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e, how to efficiently improve the mRNA payload in LNP systems is also crucial and challenging for mRNA vaccines (and other mRNA therapeutics)\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMetal ions have been reported to assemble with short nucleic acids to form nanoparticles with high- nucleic acid loading content\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e, which inspired us to apply this strategy in enriching mRNA in vaccines. As the proposed procedures shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, 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.\u003c/p\u003e \u003cp\u003eHerein, we developed a highly efficient mRNA enrichment strategy to improve the mRNA loading in lipid-based vaccine. In brief, we explored several commonly used metal ions (Fe\u003csup\u003e2+\u003c/sup\u003e, Cu\u003csup\u003e2+\u003c/sup\u003e, Zn\u003csup\u003e2+\u003c/sup\u003e and Mn\u003csup\u003e2+\u003c/sup\u003e) to prepare the M-mRNA complexes, and found that Mn\u003csup\u003e2+\u003c/sup\u003e could enrich mRNA (termed Mn-mRNA nanoparticles) in high efficiency without destroying the mRNA activity. Mn-mRNA was subsequently coated with lipids, leading to nanosystems (L@Mn-mRNA) with an over 2-fold 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.\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eDesign and screening of high-mRNA-loading nanoparticles\u003c/h2\u003e \u003cp\u003eTo 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.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The reported nanoscale complexes made of metal ions and short nucleic acids were typically prepared at 95\u0026deg;C for over 30 minutes\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. 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 minutes at 95\u0026deg;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, \u003cb\u003eFigure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e), and the expression efficiency of mEGFP and mLuc in DC 2.4 cells (DCs) (transfected by Lipofectamine\u0026trade; 3000) was significantly reduced after heating (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, D). 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\u0026deg;C, in which condition, the mRNA degradation and activity loss was significantly reduced, as demonstrated by the agarose gel (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, \u003cb\u003eFigure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e) and DCs transfection experiments (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF, G). Notably, over 95% of mRNAs (mEGFP, mLuc) maintained their activity within 5 minutes at 65\u0026deg;C. Thus, we explored the potential for enriching mRNA under this condition.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eVarious metal ions were tested to assemble with mRNA at 65\u0026deg;C. Among the Fe-mRNA, Cu-mRNA, Zn-mRNA and Mn-mRNA complexes, only Mn\u0026sup2;⁺ and Zn\u0026sup2;⁺ groups could generate regular nanostructures (\u003cb\u003eFigure S2\u003c/b\u003e). Then we do the centrifugation to collect the Mn-mRNA and Zn-mRNA nanoparticles to quantified the mRNA coordination ratio using a Quant-it\u0026trade; RiboGreen RNA Assay Kit\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. The results revealed that about 90% of mRNA formed nanoparticles in Mn-mRNA group within 5 minutes (\u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e), whereas Zn-mRNA group required 15\u0026ndash;30 minutes to reach similar efficiency (\u003cb\u003eTable S2\u003c/b\u003e). Considering the risk of mRNA degradation for prolonged treatment, we decided to use Mn\u003csup\u003e2+\u003c/sup\u003e to enrich mRNAs at 65\u0026deg;C for 5 minutes.\u003c/p\u003e \u003cp\u003eTo investigate the mechanism of metal ions-mRNA complexes formation, we calculated the coordination bond lengths (\u003cem\u003el\u003c/em\u003e) and binding energy (\u003cem\u003eΔE\u003c/em\u003e) of metal ion with the adenine (A) and guanine (G) bases, which can in some extent reflect the stability of M-mRNA complex\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. The selected sites were where transition metal ions most readily bind with bases N7/O6 (for G) and N7 (for A)\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH, Mn\u003csup\u003e2+\u003c/sup\u003e and Zn\u003csup\u003e2+\u003c/sup\u003e groups have longer bond lengths (no matter the \u003cem\u003el1\u003c/em\u003e, \u003cem\u003el2\u003c/em\u003e and \u003cem\u003el3\u003c/em\u003e) than that of Fe\u003csup\u003e2+\u003c/sup\u003e and Cu\u003csup\u003e2+\u003c/sup\u003e groups, and Mn\u003csup\u003e2+\u003c/sup\u003e 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 Fe\u003csup\u003e2+\u003c/sup\u003e and Cu\u003csup\u003e2+\u003c/sup\u003e groups did not form nanoparticles). We supposed that the formation of M-mRNA nanoparticles involved two steps: 1) metal ions coordination with mRNA bases at room temperature, potentially forming complexes without regular morphologies; 2) the heating provided energy to break the metal ion-base coordination, leading to the reassembly of complexes and form nanoparticles (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI). Thus, longer bond lengths (Mn\u003csup\u003e2+\u003c/sup\u003e and Zn\u003csup\u003e2+\u003c/sup\u003e with bases) and lower binding energy (Mn\u003csup\u003e2+\u003c/sup\u003e with bases) indicated that lower temperature could realize the reassembly. To verify our hypothesis, we analyzed the element distributions of Mn and phosphorus (P) element during assembly of Mn-mRNA by using elemental analysis (EDS) mode of transmission electron microscopy (TEM). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ and \u003cb\u003eFigure S3\u003c/b\u003e, Mn and P element had been co-localized in a disordered form before heating, and the disordered complexes reassembled into spherical nanoparticles after heating. This observation supported our proposed mechanism of the M-mRNA formation (as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI).\u003c/p\u003e \u003cp\u003eTo investigate the relationship of Mn\u003csup\u003e2+\u003c/sup\u003e 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 Mn\u003csup\u003e2+\u003c/sup\u003e to mRNA base was controlled at 8:1 to 2:1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK, \u003cb\u003eFigure S4\u003c/b\u003e, \u003cb\u003eTable S3\u003c/b\u003e), which was consistent with previous reports\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Neither increasing or decreasing Mn\u003csup\u003e2+\u003c/sup\u003e ratio (e.g. Mn\u003csup\u003e2+\u003c/sup\u003e to bases molar ratio of 20:1 or 1:1) was benefit for the nanoparticle formation. Considering that the 5:1 ratio group had lower PDI and around 100 nm in size (which are more conducive to cellular uptake)\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e, we prepared Mn-mRNA nanoparticles by using this input ratio in the following investigations.\u003c/p\u003e \u003cp\u003eTo evaluate the availability of Mn\u003csup\u003e2+\u003c/sup\u003e and mRNA during the preparation of Mn-mRNA nanoparticles, we quantitatively analyzed the percentage of input Mn\u003csup\u003e2+\u003c/sup\u003e in Mn-mRNA nanoparticles by using inductively coupled plasma mass spectrometry (ICP-MS). The result showed that 5% of the input Mn\u003csup\u003e2+\u003c/sup\u003e 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\u0026trade; 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 \u003cb\u003eMethod Section and Table S4\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eTo verify the universality of the preparation conditions (65\u0026deg;C heating for 5 minutes with 5:1 molar ratio of input Mn\u003csup\u003e2+\u003c/sup\u003e to bases), we respectively prepared Mn-mRNA with mSpike (4k nt), mLuc (1.9k nt), mOVA (1.6k nt) and mEGFP (1k nt). All groups could form regular nanoparticles (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eL, \u003cb\u003eTable S5\u003c/b\u003e), indicative of the universality of this approach.\u003c/p\u003e \u003cp\u003eSubsequently, we coated the Mn-mRNA with lipids (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) 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\u0026thinsp;\u0026plusmn;\u0026thinsp;2.4 mV) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Further increasing the lipids ratio (e.g. 20:1) did not result in the further increase in zeta potential (13.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 mV), while the polydispersity index (PDI) increased at ratios greater than 10:1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). 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.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Further increasing the lipids ratio did not significantly improve the transfection efficiency (relative mLuc expression: 1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13 vs. 1\u0026thinsp;\u0026plusmn;\u0026thinsp;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, in which case, this formulation achieved a 2-fold increase (10:1 vs. 20:1) in mRNA loading compared to current commercial products (Moderna mRNA-1273 COVID-19 vaccine) (\u003cb\u003eTable S6\u003c/b\u003e)\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCryo-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.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Four types of mRNA (mSpike, mLuc, mOVA and mEGFP) and two types of 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.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, \u003cb\u003eFigure S5\u003c/b\u003e). 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.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, \u003cb\u003eTable S5\u003c/b\u003e, \u003cb\u003eTable S7\u003c/b\u003e). 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.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). This result further demonstrated that the Mn-mRNA core had been coated by lipids.\u003c/p\u003e \u003cp\u003eThe size, PDI and zeta potential of L@Mn-mRNA did not have significant changes when incubated with PBS for seven days (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG, \u003cb\u003eFigure S6\u003c/b\u003e), indicating a good stability. Meanwhile, we also prepared LNP-mRNA according to the commercial formulation as the control group for subsequent experiments\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, and their morphology, size, zeta potential, and mRNA encapsulation efficiency were shown in \u003cb\u003eFigure S7\u003c/b\u003e and \u003cb\u003eTable S8\u003c/b\u003e. Besides, the cytotoxicity of L@Mn-mRNA and LNP-mRNA was evaluated on DCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH). At the same mRNA dosage, the lipids content in L@Mn-mRNA is approximately half that of LNP-mRNA, in which test, the concentration group (10 \u0026micro;g 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. 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 Mn\u003csup\u003e2+\u003c/sup\u003e in L@Mn-mRNA did not induce cytotoxicity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eEnhanced cellular uptake of L@Mn-mRNA by dendritic cells\u003c/h2\u003e \u003cp\u003eThen 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 and 10 hours \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, \u003cb\u003eFigure S8A\u003c/b\u003e) 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 two-fold of that in LNP-Cy5-mRNA treated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, C, \u003cb\u003eFigure S9A, B\u003c/b\u003e). 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 hours and 10 hours, respectively. Both CLSM and flow cytometry showed similar Cy5 signal intensity in each group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD-F, \u003cb\u003eFigure S8B, Figure S9C, D\u003c/b\u003e). These results suggested that the cellular uptake efficiency of L@Mn-mRNA was about 2-fold of LNP-mRNA at the same mRNA dosage.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe deduced that the enhanced cellular uptake was attributed to the core-shell nanostructure, which resulted in an increased stiffness of nanoparticles\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Therefore, we used atomic force microscopy (AFM) in mechanical mode to measure the Young\u0026rsquo;s modulus of LNP-mRNA and L@Mn-mRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). After randomly selecting twenty particles, we found that the Young\u0026rsquo;s modulus of L@Mn-mRNA was about 1.3 times higher than that of LNP-mRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH). This result supported our hypothesis that increased stiffness enhanced the cellular uptake.\u003c/p\u003e \u003cp\u003eAdditionally, confocal microscopy results showed that the colocalization of Cy5 with lysosomes (lysotracker green) decreased after 10 hours of incubation with the cells (\u003cb\u003eFigure S8A, B\u003c/b\u003e), 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 (\u003cb\u003eFigure S10\u003c/b\u003e). Notably, since the Mn-mRNA core could dissociate under physiological conditions (at PBS or FBS) (\u003cb\u003eFigure S11\u003c/b\u003e), the mRNA expression would not be affected after the lysosomal escape.\u003c/p\u003e \u003cp\u003e \u003cb\u003eL@Mn-mRNA promoted maturation and antigen presentation of dendritic cells\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e\u003c/p\u003e \u003cp\u003eBefore we investigated immune effect of L@Mn-mRNA vaccine platform, we used mLuc as the reporter mRNA to compare the \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e 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 hours, and quantified bioluminescence intensity at each time point using a plate reader. The result showed that L@Mn-mLuc had nearly two times higher \u003cem\u003ein vitro\u003c/em\u003e expression efficiency than LNP-mLuc (\u003cb\u003eFigure S12\u003c/b\u003e). We subcutaneously injected L@Mn-mLuc and LNP-mLuc into the tail bases of C57BL/6 mice, followed by bioluminescence imaging using the \u003cem\u003ein vivo\u003c/em\u003e imaging system (IVIS) at 6 and 24 h post-injection (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The images indicated that L@Mn-mLuc group had 4.1 times higher expression efficiency \u003cem\u003ein vivo\u003c/em\u003e than LNP-mLuc at 6 h and 2-fold of LNP-mLuc at 24 h (\u003cb\u003eFigure S13\u003c/b\u003e), probably due to its superior capability entering cells. We also do the subcutaneous injections on both sides of the groin, and the bioluminescence signal (\u003cb\u003eFigure S14A\u003c/b\u003e) had a similar trend with \u003cb\u003eFigure S13\u003c/b\u003e. 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 (\u003cb\u003eFigure S14B\u003c/b\u003e). Overall, due to the improved cellular uptake, the L@Mn-mRNA significantly enhanced the mRNA expression efficiency both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e, validating its potential to be an efficient mRNA delivery platform.\u003c/p\u003e \u003cp\u003eThen the capability of L@Mn-mOVA to activate the dendritic cells maturation and antigen presentation was evaluated \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. BMDCs were respectively treated with PBS, LNP-mOVA, L@Mn-mOVA (half dosage) and L@Mn-mOVA. After 24-hour incubation, the dendritic cells maturation markers, such as CD80, CD86, CD40, were detected by using flow cytometry (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB-D), and the secretion of pro-inflammatory cytokines IL-6 and TNF-α in the cell culture medium was tested by ELISA kits (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE \u003cb\u003eand F\u003c/b\u003e). The results showed that the L@Mn-mOVA group was 1.2-fold expression of CD80, 1.2-fold expression of CD86, and 1.5-fold expression of CD40 compared to the LNP-mOVA (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB-D). The IL-6 and TNF-α secretion was both approximately 3-fold of that in the LNP-mOVA group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE, 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.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). Notably, even the L@Mn-mOVA (half dosage) group showed significant increases in these results compared to the LNP-mOVA group.\u003c/p\u003e \u003cp\u003eWe also explored the potential immune response enhancement caused by Mn\u003csup\u003e2+\u003c/sup\u003e, because Mn\u003csup\u003e2+\u003c/sup\u003e can be act as an agonist of cGAS-STING pathway\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e,\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. However, we did not observe significant increase of the STING protein expression in BMDCs (\u003cb\u003eFigure S15\u003c/b\u003e) after incubation with L@Mn-mOVA, which probably because that the small amount (0.4% in the whole formulation) Mn\u003csup\u003e2+\u003c/sup\u003e could not activate the cGAS-STING pathway. Therefore, the enhanced immune response in the L@Mn-mOVA group was more likely attributed to high mRNA loading and improved cellular uptake.\u003c/p\u003e \u003cp\u003eTo evaluate \u003cem\u003ein vivo\u003c/em\u003e responses, each formulation (PBS, LNP-mOVA, L@Mn-mOVA with different dosages) was subcutaneously injected into the inguinal region of C57BL/6 mice. Inguinal lymph nodes were harvested for flow cytometric analysis at the 24 h time point after administration. with the results showing that L@Mn-mOVA group exhibited 3.5-fold expression of CD80, 4.5-fold expression of CD86, and 4.1-fold expression of CD40 compared to the group treated with LNP-mOVA (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH-J). As for the \u003cem\u003ein vivo\u003c/em\u003e antigen presentation, the L@Mn-mOVA group achieved 2.6-fold SIINFEKL-H-2Kb-positive cells compared to the LNP-mOVA group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eK). 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\u0026thinsp;+\u0026thinsp;and SIINFEKL-H-2Kb-positive cells) were higher than that of LNP-mOVA. The gating strategy of BMDCs maturation and antigen presentation are presented in \u003cb\u003eFigure S16\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eTherapeutic efficacy of L@Mn-mRNA vaccine in B16-OVA melanoma tumour model\u003c/h2\u003e \u003cp\u003eTo evaluate the anti-tumour efficacy of L@Mn-mRNA vaccine, the B16-OVA melanoma tumour model was established on C57BL/6 mice, and the mice were subcutaneously administered in the inguinal regions with PBS, LNP-mOVA (10 \u0026micro;g, representing 10 \u0026micro;g mOVA per mouse), L@Mn-mOVA (5 \u0026micro;g, representing half dosage 5 \u0026micro;g mOVA per mouse) and L@Mn-mOVA (10 \u0026micro;g, representing 10 \u0026micro;g mOVA per mouse) three times (on Day 4, 7, and 10) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). The L@Mn-mOVA group exhibited higher efficiency on tumour-suppression than that of the LNP-mOVA group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, \u003cb\u003eFigure S17A\u003c/b\u003e), with improving median survival rate by 26% (46\u0026ndash;58 days) (\u003cb\u003eFigure S17B\u003c/b\u003e). The half dosage L@Mn-mOVA still exhibited a certain degree of superior anti-tumour effect compared to the LNP-mOVA group, extending median survival by 13% (46\u0026ndash;52 days) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, \u003cb\u003eFigure S17B\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eThen we evaluated the antigen-specific immune response in blood and splenocytes. The L@Mn-mOVA (10 \u0026micro;g) group showed 8-fold increase in the proportion of SIINFEKL tetramer\u0026thinsp;+\u0026thinsp;cells within CD8\u0026thinsp;+\u0026thinsp;T cells in the blood and 1.8-fold increase in the spleen compared to the LNP-mOVA group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, D). Although the difference between the L@Mn-mOVA (5 \u0026micro;g) group and the LNP-mOVA group in the blood was negligible, the L@Mn-mOVA (5 \u0026micro;g) group showed a significant increase in SIINFEKL tetramer\u0026thinsp;+\u0026thinsp;cells within CD8\u0026thinsp;+\u0026thinsp;T cells compared to the LNP-mOVA group in the spleen (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, D). Further, splenocytes isolated from mice subjected to various treatment groups were analyzed thourogh the IFN-ELISpot assay. The half-dose L@Mn-mOVA (5 \u0026micro;g) group and the L@Mn-mOVA group (10 \u0026micro;g) exhibited 3-fold and 3.5-fold increases in the number of spots compared to the LNP-mOVA group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE, F), respectively. There results demonstrated that the L@Mn-mOVA groups (including the half-dose group) effectively activated antigen-specific immune responses.\u003c/p\u003e \u003cp\u003eFor 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.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG-J), and the TNF-α and IFN-β in L@Mn-mOVA (5 \u0026micro;g) group were also higher than that of the LNP-mOVA group, indicating an enhanced immunogenic effectiveness.\u003c/p\u003e \u003cp\u003eThen the immune infiltration at the tumour site was assessed by flow cytometry. The L@Mn-mOVA (10 \u0026micro;g) group exhibited 2-fold ratio of CD3\u0026thinsp;+\u0026thinsp;CD8\u0026thinsp;+\u0026thinsp;T cells and 1.8-fold ratio of CD3\u0026thinsp;+\u0026thinsp;CD4\u0026thinsp;+\u0026thinsp;T cells compared to that of LNP-mOVA group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eK, L). The L@Mn-mOVA (10 \u0026micro;g) group also exhibited 1.5-fold number of CD11c\u0026thinsp;+\u0026thinsp;cells (antigen-presenting cells) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eM). The L@Mn-mOVA (5 \u0026micro;g) group showed a significant increase in the number of CD3\u0026thinsp;+\u0026thinsp;CD8\u0026thinsp;+\u0026thinsp;T cells, CD3\u0026thinsp;+\u0026thinsp;CD4\u0026thinsp;+\u0026thinsp;T cells and similar number of CD11c\u0026thinsp;+\u0026thinsp;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 tumours. Additionally, the half-dose group, L@Mn-mOVA (5 \u0026micro;g) showed comparable levels of immune activation to LNP-mOVA group.\u003c/p\u003e \u003cp\u003eTo 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 tumour inoculation, and the mice were sacrificed for analysis on Day 70 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eN). At the same dosage, the L@Mn-mOVA group had a higher proportion of CD44\u003csup\u003ehigh\u003c/sup\u003eCD62L\u003csup\u003ehigh\u003c/sup\u003e T cells (central memory T cells) and CD44\u003csup\u003ehigh\u003c/sup\u003eCD62L\u003csup\u003elow\u003c/sup\u003e T cells (effector memory T cells) in the spleen compared to the LNP-mOVA group, indicating stronger immune memory (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eO, P). The gating strategy of central memory T cells and effector memory T cells was presented in \u003cb\u003eFigure S18\u003c/b\u003e. Based on these results, the L@Mn-mOVA exhibited superior anti-tumour efficacy compared to LNP-mOVA at the same dosage. Even the L@Mn-mOVA (5 \u0026micro;g) group, which meant the lipid usage was a quarter of that in LNP-mOVA, achieved effective anti-tumour immune response.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eBioeffects and safety evaluation of L@Mn-mRNA vaccine\u003c/h2\u003e \u003cp\u003eIt is crucial to evaluate the safety of our L@Mn-mRNA vaccine platform. During the therapeutic period, no fluctuations were observed in the body weight of the mice (\u003cb\u003eFigure S19\u003c/b\u003e), 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 (\u003cb\u003eFigure S20\u003c/b\u003e). 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 (\u003cb\u003eFigure S21\u003c/b\u003e), which indicated the formulations did not affect the functions of major organs.\u003c/p\u003e \u003cp\u003eMeanwhile, 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 and Anti-PEG IgG antibody titers measurement was presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA. In the PBS group, Anti-PEG IgM levels showed no significant change before and after administration. In the LNP-mRNA (10 \u0026micro;g) 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 10-fold increase in anti-PEG IgM level in L@Mn-mRNA (10 \u0026micro;g) group and only 40% of the mice had a 5-fold increase in L@Mn-mRNA (5 \u0026micro;g) group. Similarly, as for the measurement of Anti-PEG IgG, in the LNP-mRNA (10 \u0026micro;g) group, 80% of the mice exhibited a substantial increase (ranging from a minimum of 6-fold to a maximum of 9-fold). In the L@Mn-mRNA (10 \u0026micro;g) group, only 20% of the mice exhibited a notable elevation in antibody levels (9-fold), with the remaining mice displaying marginal increments. In the L@Mn-mRNA (5 \u0026micro;g) group, all mice had no significant increase (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). 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 (ADA), which potentially offered an effective strategy to avoid the accelerated blood clearance.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, we have developed a platform technique for improving mRNA loading in lipid-based nanoparticles. Mn\u003csup\u003e2+\u003c/sup\u003e could 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. The platform technique could be suitable for types of novel ionizable lipids and mRNA. Thus, this work holds the potential to provide references for the next generation of lipid-based mRNA vaccines.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eMaterials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll 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\u0026nbsp;(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), 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 (PMSF) was purchased from Beijing Lablead. D-Luciferin (D1007) was purchased from Solarbio Biotech Co., Ltd. STING (D2P2F) Rabbit mAb (13647) and Anti-rabbit IgG, HRP-linked Antibody 7074 (Goat) were purchased from Cell Signaling Technology (CST). RPMI 1640 medium and Opti-MEM reduced serum medium were obtained from Thermo Fisher Scientific Inc.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMn-mRNA and Lipid@Mn-mRNA (L@Mn-mRNA) preparation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor a reaction volume of 200 μL, we selected a 600 μL EP tube. First, 50 μL of 6 mM Mn\u003csup\u003e2+\u003c/sup\u003e 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 minutes. Afterwards, the sample was heated in the metal bath at 65 °C for 5 minutes. After completion of the reaction, the sample was cooled to the room temperature. The Mn-mRNA nanoparticles were collected through centrifugation at 60000 g for 15 minutes, and the Mn-mRNA nanoparticles are dispersed in 200 μL of enzyme-free water. The mRNA and Mn\u003csup\u003e2+\u003c/sup\u003e 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 hour. 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.\u003c/p\u003e\n\u003cp\u003eFor the preparation of Fe-mRNA, Cu-mRNA, and Zn-mRNA, 6 mM M\u003csup\u003e2+\u003c/sup\u003e solutions were respectively prepared using ferric chloride tetrahydrate, anhydrous copper (II) sulfate, and zinc nitrate enneahydrate. The ratios of M\u003csup\u003e2+\u003c/sup\u003e to bases were referred from the literature\u003csup\u003e33,34\u003c/sup\u003e. The remaining steps were identical to the preparation method of Mn-mRNA. When preparing Mn-mRNA with different Mn\u003csup\u003e2+\u003c/sup\u003e 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMn-mRNA, L@Mn-mRNA and LNP-mRNA characterization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 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 dynamic light scattering (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 3\u003csup\u003e36\u003c/sup\u003e. The mRNA content in both particles and supernatant was measured separately using the Quant-it™ RiboGreen RNA Assay Kit (n = 3 in each group). The Mn\u003csup\u003e2+\u003c/sup\u003e 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. Three independent replicate experiments were performed.\u003c/p\u003e\n\u003cp\u003eThe quantitative calculation of the Mn-mRNA composition was showed below:\u003c/p\u003e\n\u003cp\u003e1. Mn ratio involved in the reaction (by ICP-MS) equals to\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e“Mn in the precipitate (Mn-mRNA) (μg)” / “Mn in the precipitate (Mn-mRNA) (μg) + Mn in the supernatant solution (μg)”\u003c/p\u003e\n\u003cp\u003e2. Mn amount involved in the reaction equals to “Mn Input” × “Mn ratio involved in the reaction”\u003c/p\u003e\n\u003cp\u003e3. mRNA amount involved in the reaction equals to “mRNA Input” × “mRNA encapsulation efficiency”\u003c/p\u003e\n\u003cp\u003e4. The proportion of mRNA in Mn-mRNA equals to “mRNA involved (μg)” / “Mn involved (μg) + mRNA involved (μg)”\u003c/p\u003e\n\u003cp\u003e5. The proportion of Mn in Mn-mRNA equals to “Mn involved (μg)” / “Mn involved (μg) + mRNA involved (μg)”\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAgarose gel electrophoresis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAgarose gel electrophoresis was run on a 1 % agarose gel using 1X TAE as electrophoresis buffer at 100 V for 30 minutes. The mRNA sample amount in each well was 0.5 μg. Subsequently, we utilized ultraviolet light for imaging.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSimulated calculation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe geometric and electronic properties of metal ions-mRNA have been computed using the Gaussian 09W program package at B3LYP/6-31G(d,p) level\u003csup\u003e47\u003c/sup\u003e. 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 Mutifwn\u003csup\u003e48\u003c/sup\u003e. Utilizing the iefpcm implicit solvent model, single-point energy calculations for the structures were performed employing the wb97xd\u003csup\u003e49\u003c/sup\u003e functional with the def2TZVP\u003csup\u003e50\u003c/sup\u003e basis set. Frequency calculations were carried out using the 6-311Gd\u003csup\u003e51\u003c/sup\u003e basis set to determine the binding energies of the complexes at 298K for different components.\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"data:image/png;base64,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\"\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLNP-mRNA and L@Mn-mRNA Young’s moduli test from AFM\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInitially, 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 minutes. 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.\u0026nbsp;Seven data points were selected for quantitative analysis in each group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCells and animals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFemale C57BL/6 (6–8 weeks old) were purchased from SPF Biotechnology. \u0026nbsp;This study was approved by the ethics committee of the National Center for Nanoscience and Technology of China (approval number NCNST21-2103-0401). 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 (ATCC, 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 ℃ with 5% CO\u003csub\u003e2\u003c/sub\u003e. Bone Marrow-Derived Dendritic Cells\u003cstrong\u003e\u0026nbsp;(\u003c/strong\u003eBMDCs\u003cstrong\u003e)\u0026nbsp;\u003c/strong\u003ewere 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 minutes 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 minutes. 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.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eOn Day 6, the nonadherent cells were aspirated and incubated in 6-well plates with fresh medium for further investigation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIn vitro\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;transfection\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDC 2.4 cells were seeded in a 96-well plate at a density of 10\u003csup\u003e4\u003c/sup\u003e 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 hours, 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.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCellular uptake and lysosomal escape\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eL@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 hours. DC 2.4 cells were stained with Hoechst 33342 (1:500) for 5 minutes 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 minutes 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).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDC 2.4 cells were seeded in a 12-well plate at a density of 10\u003csup\u003e5\u003c/sup\u003e 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 10000 live cells in APC channel (n = 5 in each group).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIn vitro\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;and \u003cem\u003ein vivo\u003c/em\u003e Bioluminescence\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eL@Mn-mLuc and LNP-mLuc were respectively incubated in BMDCs for 12 and 24 hours. 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.\u003c/p\u003e\n\u003cp\u003eAt 6 and 24 hours 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 \u003cem\u003ein vivo\u003c/em\u003e imaging system (PerkinElmer IVIS). Bioluminescence was quantified using the Living Image software (PerkinElmer).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blot analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eβ-actin and STING protein expression in BMDCs were analyzed by western blot. 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*10\u003csup\u003e5\u003c/sup\u003e cells per well) for 24 hours. Subsequently, cells were lysed on ice using cell lysis buffer containing protease and phosphatase inhibitors. The lysate was then centrifuged in a pre-chilled centrifuge. Centrifugation was conducted at 4 °C and 12,000 rpm for 20 minutes. After centrifugation, the supernatant was collected for protein quantification. The protein concentration of the samples was quantified using a bicinchoninic acid assay (BCA protein assay kit; Beyotime) according to the manufacturer’s instructions. Protein lysates (15 μg per sample) were resolved by SDS-PAGE and transferred to a polyvinylidene fluoride membrane. After being blocked in 5% BSA, the polyvinylidene fluoride membrane was incubated with primary antibodies followed by the specific secondary antibodies. Immunoreactive proteins were visualized using enhanced chemiluminescence reagents (Bio-Rad). Quantitively analysis of WB results were carried out using ImageJ software (n = 3 in each group).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEnzyme-linked immunosorbent assay (ELISA)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePBS, 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*10\u003csup\u003e5\u003c/sup\u003e cells per well) for 24 hours. The supernatant was collected for the detection of IL-6 and TNF-α secretion from BMDCs. After diluting the supernatant five-fold, 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.\u003c/p\u003e\n\u003cp\u003eFor 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 five-fold. 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).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eL@Mn-mRNA promoted maturation and antigen presentation of DCs \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBMDCs were seeded into a 12-well plate (1 × 10\u003csup\u003e5\u003c/sup\u003e 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 hours. The medium from each group was collected for ELISA of TNF-α, IL-6, IFN-1α and IFN-1β 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 minutes at 4 °C before the detection of flow cytometry (ACEA NovoExpress).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eiLN from each mouse was harvested at 24 hours 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), APC 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).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnti-tumour effects in a subcutaneous B16-OVA cancer model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate the anti-tumour effect of the L@Mn-mRNA vaccine in a solid tumour model, 1 × 10\u003csup\u003e6\u003c/sup\u003e murine melanoma cells B16-OVA cells were injected subcutaneously into the right flank of C57BL/6 mice on Day 0. The mice 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 4, 7 and 10. Mice treated with PBS were used as a negative control group. The tumour volume was measured every other day using Vernier calipers and calculated by the following formula: tumour volume = length × 1/2 width\u003csup\u003e2\u003c/sup\u003e. The mice were euthanized on Day 17. The tumours were collected, and digested into single-cell suspensions to analyze the infiltrating immune cells by flow cytometry. The resulting cell suspensions were collected and stained with FITC anti-mouse CD3 antibody (1:100), PE-Cy7 anti-mouse CD8a antibody (1:100), APC anti-mouse CD4 antibody (1:100), PE anti-mouse F4/80 antibody (1:100) and APC anti-mouse SIINFEKL-H-2kb antibody (1:100) for 30 minutes at 4 °C before being analyzed by flow cytometry (ACEA NovoExpress). Blood and spleens were collected to analyze the proportion of specifical T cells that recognize the SIINFEKL antigen. \u0026nbsp; Spleens were collected for IFN-γ ELISpot analysis. According to the manufacturer’s instructions, splenocytes were seeded in a 96 well plate (10\u003csup\u003e5\u003c/sup\u003e cells per well), pre-coated with a mouse anti-IFN-γ antibody and incubated with SIINFEKL peptide for 20 hours. A biotinylated antibody specific for IFN-γ and alkaline-phosphatase conjugated to streptavidin were subsequently used to detect the IFN-γ secreted by the re-stimulated T cells. By adding a substrate solution, visual spots were formed at the sites of captured IFN-γ, and automated spot quantification was caried out by Dakewe Biotech. Blood and the major organs, including heart, liver, spleen, lung and kidney, were collected for hematoxylin and eosin (H\u0026amp;E) staining to assess the safety of the L@Mn-mRNA vaccines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLong-term immune memory test\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 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, PE-Cy7 anti-mouse CD8, APC anti-mouse CD44 and PE anti-mouse CD62L antibodies. The central memory T cells (CD3+CD8+CD44\u003csup\u003ehigh\u003c/sup\u003eCD62L\u003csup\u003ehigh\u003c/sup\u003e) and effector memory T cells (CD3+CD8+CD44\u003csup\u003ehigh\u003c/sup\u003eCD62L\u003csup\u003elow\u003c/sup\u003e) were analyzed by flow cytometry (ACEA NovoExpress).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnti-PEG IgG and IgM measurement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 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 minutes. Finally, 100 μL stop solution was added, and the results were collected at a plate reader.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll 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. \u003cem\u003eP\u003c/em\u003e values of *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, and ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001 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).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis 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). \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe sincerely acknowledge the valuable discussions with Ms. Yike Wu, Dr. Na Chen, Dr. Shuai Liu, Dr. Guangna Liu and Dr. Keman Cheng. Elements of Figure 1, 2 and 3 were created with BioRender.com and had got the publication license.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eX.M., T.J. and G.N. conceived the idea, analyzed the data, and wrote the manuscript. X.M., S.L., A.L., S.Z., Z.L., M.Z., Z.L., J.Z., J.L., Q.C., W.L. performed experiments. H.Q., J.L. and X.L. provided advice on \u003cem\u003ein vivo\u003c/em\u003e experiments. X.S. and H.W. supervised and conducted the computational simulation portion. X.Z. and J.L. 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.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eFang, E. \u003cem\u003eet al.\u003c/em\u003e Advances in COVID-19 mRNA vaccine development. 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The Journal of chemical physics 89, 2193\u0026ndash;2218 (1988).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"manganese ions-mediated mRNA enrichment, improved mRNA loading, reduced lipids usage, mRNA vaccine platform, enhanced immune response","lastPublishedDoi":"10.21203/rs.3.rs-4755456/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4755456/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eLipid 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 efficiency of mRNA in LNPs not only compromises the vaccine\u0026rsquo;s efficacy but also heightens the risk of non-specific immune responses, accelerates clearance from the bloodstream, and exacerbates side effects associated with the lipid carriers. These problems underscore the urgent need for improving mRNA loading in LNPs to provide dose-sparing effects. Herein, we developed a manganese ion (Mn\u0026sup2;⁺) mediated mRNA enrichment strategy to efficiently form a high-density mRNA core, termed Mn-mRNA nanoparticle, which is subsequently coated with lipids. The resulting nanosystem, L@Mn-mRNA, achieved over twice the mRNA loading compared to conventional mRNA vaccine formulations (LNP-mRNA). Remarkably, L@Mn-mRNA also demonstrated 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 achieved significantly enhanced antigen-specific immune responses and therapeutic efficacy as vaccines. We elucidated the mechanism behind Mn-mRNA construction and optimized the L@Mn-mRNA formulations, and this method is suitable for types of lipids and mRNAs. Thus, this strategy holds significant potential as a platform for the next generation of lipid-based mRNA vaccines.\u003c/p\u003e","manuscriptTitle":"Engineering of mRNA vaccine platform with reduced lipids and enhanced efficacy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-08 02:16:45","doi":"10.21203/rs.3.rs-4755456/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"83d8d98e-124e-4561-8559-a713c5f51b96","owner":[],"postedDate":"August 8th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":35673739,"name":"Biological sciences/Biotechnology/Nanobiotechnology/Nanoparticles"},{"id":35673740,"name":"Physical sciences/Nanoscience and technology/Nanomedicine/Drug delivery"}],"tags":[],"updatedAt":"2024-10-15T11:05:38+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-08 02:16:45","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4755456","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4755456","identity":"rs-4755456","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}