Lcaps, methods of making and using the same

Abstract

The application provides a Lycopene Acidic Polysaccharide Lipid Nanoparticle (LCAP LNP) and a preparation method and application thereof, the preparation method of the Lycopene Acidic Polysaccharide Lipid Nanoparticle (LCAP LNP) comprises the following steps: dissolving Lycopene Acidic Polysaccharide (LCAP) in water as an aqueous phase; dissolving ionizable cationic lipids, auxiliary phospholipids, cholesterol and polyethylene glycol lipids in 95% ethanol in proportion as an alcohol phase; mixing the aqueous phase and the alcohol phase through a microfluidic technology to form nanoparticles; performing ultrafiltration purification on the obtained nanoparticles, and replacing the outer phase with a Tris-HCl buffer solution, so that the Lycopene Acidic Polysaccharide Lipid Nanoparticle (LCAP LNP) is obtained. The application constructs a brand-new "polysaccharide-LNP" composite adjuvant system, and provides a brand-new solution for the development of a new generation of efficient, safe and multifunctional vaccine adjuvant.

Description

Technical Field

[0001] This invention belongs to the field of vaccine technology, specifically relating to a Lysimachia christinae acidic polysaccharide lipid nanoparticle (LCAP / LNP), its preparation method, and its application. Background Technology

[0002] Vaccines are one of the most effective and cost-effective public health tools for preventing and controlling infectious diseases. However, due to factors such as pathogen mutation, immune escape, and population immune variability, relying solely on the antigen itself often fails to achieve an ideal protective immune response with lower doses or fewer immunizations. Novel vaccine platforms, represented by recombinant subunit proteins, synthetic peptides, and nucleic acids (such as mRNA and DNA), have become the mainstream direction in current vaccine development due to their advantages such as high safety, flexible design, and rapid production.

[0003] Meanwhile, lipid nanoparticles (LNPs) have become one of the most successful nucleic acid drug delivery platforms, particularly achieving revolutionary success in the field of mRNA vaccines. Their excellence lies in their ability to efficiently encapsulate and protect nucleic acids, and to deliver them efficiently to the cytoplasm through endocytosis / endosome escape mechanisms. LNPs consist of ionizable cationic lipids, helper phospholipids, cholesterol, and PEGylated lipids. The ionizable cationic lipids are positively charged in acidic preparation environments, allowing them to bind tightly to negatively charged nucleic acids (such as mRNA and siRNA) via electrostatic interactions, achieving efficient encapsulation. Upon entering the near-neutral pH environment in vivo, they become electrically neutral, significantly reducing toxicity. Most importantly, they promote "endosome escape"—helping nucleic acids escape into the cytoplasm before the rupture of endosome vesicles, a crucial step for nucleic acids to perform their functions (translation or gene silencing).

[0004] However, traditional LNP designs primarily focus on delivery efficiency and safety, with their function as immunostimulants being relatively limited or underdeveloped. While some adjuvant effects can be incidentally generated by modulating lipid components, this is typically a passive, non-specific immune activation. Therefore, developing safe and efficient vaccine adjuvants is crucial for the successful application of these novel vaccines. Summary of the Invention

[0005] This invention aims to provide a Lysimachia christinae acidic polysaccharide lipid nanoparticle (LCAP / LNP), its preparation method, and its application, thereby constructing a novel "polysaccharide-LNP" composite adjuvant system and providing a new solution for the development of next-generation, efficient, safe, and multifunctional vaccine adjuvants.

[0006] To achieve its technical objectives, this application adopts the following technical solution:

[0007] This invention provides a method for preparing Lysimachia christinae acidic polysaccharide lipid nanoparticles (LCAP LNP), comprising: dissolving Lysimachia christinae acidic polysaccharide (LCAP) in water as the aqueous phase; dissolving ionizable cationic lipids, cofactor phospholipids, cholesterol, and polyethylene glycol-modified lipids in 95% ethanol in a certain proportion as the alcohol phase; mixing the aqueous phase and the alcohol phase using microfluidic technology to form nanoparticles; purifying the obtained nanoparticles by ultrafiltration and replacing the outer phase with Tris-HCl buffer to obtain Lysimachia christinae acidic polysaccharide lipid nanoparticles (LCAP LNP).

[0008] Preferably, the aqueous phase further contains mRNA, and the mass ratio of LCAP to mRNA is 1:1.

[0009] Preferably, the molar ratio of the ionizable cationic lipid, cofactor phospholipid, cholesterol, and polyethylene glycol ester is 50:10:38.5:1.5.

[0010] Preferably, the ultrafiltration purification is performed using a 30 kDa filter membrane at 10°C and 4500 rpm. After concentrating to one-tenth of the original volume, Tris-HCl buffer is added to the original volume, and the operation is repeated 3 times.

[0011] The present invention also provides Lysimachia christinae acidic polysaccharide lipid nanoparticles (LCAP LNP), which are obtained by the above preparation method.

[0012] This invention also provides the application of the above-mentioned Lysimachia christinae acidic polysaccharide lipid nanoparticles (LCAP LNP) in the preparation of vaccine adjuvants.

[0013] The present invention also provides a vaccine adjuvant comprising the above-mentioned Lysimachia christinae acidic polysaccharide lipid nanoparticles LCAPLNP.

[0014] Preferably, the vaccine adjuvant further includes pharmaceutical excipients.

[0015] The present invention also provides an mRNA vaccine composition comprising a viral antigen or mRNA encoding the viral antigen, and the above-mentioned Lysimachia christinae acidic polysaccharide lipid nanoparticles LCAP LNP.

[0016] Preferably, the vaccine is a rabies virus vaccine.

[0017] The beneficial effects of this invention are as follows:

[0018] 1. This invention combines Lysimachia christinae acidic polysaccharide (LCAP) with lipid nanoparticle (LNP) system to construct a composite system with both delivery and immune enhancement effects, thereby providing a new technical solution for vaccine adjuvant development.

[0019] 2. The LCAP and LNP system described in this invention has good assembly compatibility and formulation stability, which is conducive to the formation of nanoparticles with suitable particle size, good dispersibility and stable structure, and has good formulation feasibility.

[0020] 3. The LCAP LNP prepared by this invention can promote the activation and maturation of antigen-presenting cells, which is beneficial to improving the initiation efficiency of the immune response.

[0021] 4. The LCAP LNP described in this invention can simultaneously enhance humoral immunity and cellular immunity, thereby improving the overall immune effect of the vaccine and enhancing its protective effect against lethal viral attacks in vivo.

[0022] 5. This invention improves the immune effect while exhibiting good safety and biocompatibility, thus showing promising application prospects. Attached Figure Description

[0023] Figure 1 Figure showing the particle size distribution of LNPs co-encapsulated with different polysaccharides and mRNA;

[0024] Figure 2 The graph shows the particle size detection results for LCAP and mRNA at different ratios.

[0025] Figure 3 Photographs of the appearance of G mRNA LNP, LCAP LNP and G mRNA+LCAP LNP formulations;

[0026] Figure 4 Representative transmission electron microscope (TEM) images showing the spherical morphology of nanoparticles;

[0027] Figure 5 Figure 1 shows the results of a flow cytometry experiment to assess antigen presentation capability.

[0028] Figure 6 For DC2.4 cell maturity analysis;

[0029] Figure 7 This is a diagram showing the results of a spleen lymphocyte proliferation experiment.

[0030] Figure 8 This is a graph showing the results of multiple cytokine detection.

[0031] Figure 9 The effect of LCAP on IgG antibody levels in immunized mice;

[0032] Figure 10 The effect of LCAP on neutralizing antibody levels in immunized mice;

[0033] Figure 11 For LCAP to CD4+ IFN-γ in T cell population + TNF-α + and IL-2 + The effect of cell frequency;

[0034] Figure 12 For LCAP to CD8 + IFN-γ in T cell population + TNF-α + and IL-2 + The effect of cell frequency;

[0035] Figure 13 IFN-γ co-expressed in T cell subsets + and TNF-α + Quantitative analysis of multifunctional T cells;

[0036] Figure 14 The proportion of GC B cells in the inguinal lymph nodes;

[0037] Figure 15 The proportion of follicular helper T cells (Tfh);

[0038] Figure 16 Kaplan-Meier survival curves for mice challenged with a lethal virus;

[0039] Figure 17 The changes in body weight of mice after being challenged with a lethal virus;

[0040] Figure 18 These are representative gross anatomical photographs of the major organs of the mice collected.

[0041] Figure 19 Representative sections of the heart, liver, spleen, lung, and kidney tissues stained with hematoxylin and eosin (H&E);

[0042] Figure 20 Serum biochemistry and inflammatory profile analysis. Detailed Implementation

[0043] The present invention will now be described in detail with reference to specific embodiments. The following specific embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any way.

[0044] Example 1: Preparation of G mRNA LNP, LCAP LNP and G mRNA+LCAP LNP

[0045] Prepare appropriate volumes of LNP mixtures as the aqueous phase according to the required dosage. For LCAP-only encapsulation (LCAP LNP), the aqueous phase contains LCAP dissolved in water for injection; for mRNA-only encapsulation (G mRNA LNP), the aqueous phase contains G mRNA; for co-encapsulation of LCAP and mRNA (G mRNA+LCAP LNP), LCAP and mRNA are mixed in the aqueous phase according to the specified ratio. All solutions are diluted to appropriate concentrations using citrate buffer (50 mM, pH 4.0) and then filtered through a 0.22 μm sterile filter. Ionizable cationic lipids, cofactor phospholipids, cholesterol, and polyethylene glycol-modified lipids (DMG-PEG2000) are dissolved in 95% ethanol according to the specified ratio to prepare the alcohol phase. The aqueous and alcohol phases are then mixed using microfluidic technology to prepare the LNPs. The obtained nanoparticles were purified by ultrafiltration using a 30 kDa filter membrane at 10°C and 4500 rpm. The solution was concentrated to one-tenth of the original volume, and Tris-HCl buffer (pH 7.4) was added to the original volume. The solution was then concentrated to one-tenth of the original volume again. This was recorded as one solution change, and a total of three solution changes were performed.

[0046] Example 2: Effects of different polysaccharide structures on LNP encapsulation efficiency and immunomodulatory activity

[0047] The inventors discovered in their research that the charged properties of polysaccharides are a key factor determining whether they can be efficiently co-encapsulated with mRNA within LNPs. To investigate the effects of structural differences in polysaccharides (mainly acidic / neutral) on the formation, physicochemical properties, and adjuvant activity of polysaccharide-mRNA co-encapsulated LNPs, this example selected LCAP, strongly anionic polysaccharide dextran sulfate, weakly acidic polysaccharide pectin, neutral polysaccharide β-glucan, and cationic polysaccharide chitosan. These polysaccharides were mixed with G mRNA at a mass ratio of 1:1, and co-encapsulated LNPs were prepared according to the method in Example 1. A group encapsulated with G mRNA alone was set up as a control. The particle size of the LNPs in each group was detected using dynamic light scattering (DLS). Figure 1 The polydispersity index (PDI), zeta potential, and mRNA encapsulation efficiency are shown in Table 1. The LCAP used in this invention is rich in uronic acid. In an acidic aqueous phase (pH 4.8), its carboxyl group is protonated, but it still retains a certain degree of negative charge. This moderate negative charge allows it to electrostatically compete with the similarly negatively charged mRNA, jointly binding to ionizable cationic lipids. Further research revealed that when LCAP and mRNA are mixed in a specific ratio (e.g., a mass ratio of 1:1), they form a more ordered core-shell structure within the LNP. In contrast, neutral polysaccharides, lacking charge interaction, are difficult to effectively encapsulate within the LNP; while the strongly anionic polysaccharide—glucan sulfate—may excessively compete, disrupting the integrity of the LNP structure. The cationic polysaccharide chitosan directly forms a precipitate.

[0048] Table 1 shows the PDI, Zeta potential, and encapsulation efficiency of different polysaccharides co-encapsulated with mRNA and LNP.

[0049] sulfated dextran 0.24 -14.52 86.3 LCAP 0.08 -6.60 98.74 pectin 0.15 -8.90 96.39 β-glucan 0.21 -2.60 92.5 Chitosan precipitation 21.70 precipitation

[0050] Example 3: Characterization of LCAP LNP

[0051] LCAP and mRNA were mixed in aqueous phase at mass ratios of 1:1, 1:2, 1:5, and 1:10, respectively, and the particle size was measured using a nanometer. The results showed that a 1:1 mass ratio of LCAP to G mRNA resulted in the optimal LNP particle size, approximately 100 nm. Figure 2 ).

[0052] Macroscopic observation after standing showed that all three formulations, including G mRNA LNP, LCAP LNP, and G mRNA+LCAP LNP, exhibited a uniform, translucent, milky-white appearance with no visible precipitation or aggregation. Figure 3 Transmission electron microscopy (TEM) images further confirmed the results. Figure 4 All LNPs exhibited a typical spherical morphology with a smooth surface, indicating that the co-encapsulation strategy of G mRNA and LCAP did not negatively affect the physical stability of the nanoparticles.

[0053] Example 4: Cellular effects of LCAP and LNP as vaccine adjuvants

[0054] LCAP and LNP stimulation of antigen presentation in DC2.4 cells: To evaluate the effect of LCAP-related agents on the antigen-presenting potential of DCs from the perspective of MHC I / MHC II co-expression, they were co-incubated with DC2.4 cells. DC2.4 cells were revived and cultured to the logarithmic growth phase, then passaged and seeded in 6-well plates. After acclimatization at 37°C and 5% CO2 for 24 h, the cells were divided into 5 groups: blank control group (0.01M PBS), blank LNP group, LCAP group, LCAP and LNP group, and positive control group (LPS 1 μg / mL). All treatments were adjusted to 100 μg / mL, filtered through a 0.22 μm membrane, and added at a rate of 10 μL / well. Cells were incubated for 48 h.

[0055] After treatment, cells were collected, washed twice with PBS, and counted. 2 × 10⁶ cells were then collected. 6 Cells were seeded in 96-well V-plates and incubated in the dark at room temperature using BD Horizon. TM The sample was stained with immobilizable reactive dye 780 (BD Biosciences, 565388) for 15 min, then washed twice with PBS containing 2% fetal bovine serum. Finally, it was stained with TruStain FCX. TMCells were blocked with Plus (anti-mouse CD16 / 32) antibody (BioLegend, 156603) in the dark at room temperature for 10 min, and then stained with a mixture of multiple surface antibodies: FITC anti-mouse CD11c (BioLegend, 117305), PE anti-mouse H-2kb (BioLegend, 116507), and APC anti-mouse IA / IE (BioLegend, 107613) in the dark at 4°C for 30 min. After staining, the cells were washed twice with PBS containing 2% fetal bovine serum, resuspended in PBS, filtered through a mesh, and analyzed by flow cytometry (CytoFLEX, Backman, USA). The results showed that the proportion of MHC I / II positive cells in the LCAP LNP group was approximately 2.5 times that in the PBS group, and significantly higher than that in the LCAP group alone (p<0.0001). Figure 5 This suggests that encapsulating LCAP within LNP may enhance its stimulatory effect on DC.

[0056] The maturation-promoting effect of LCAP LNP on DC2.4 cells: To verify whether LCAP LNP promotes DC cell maturation and activation, DC2.4 cells were treated according to the antigen presentation method described for LCAP LNP stimulation. After incubation for 48 hours, cells were collected, washed, and counted. 2 × 10⁶ cells were collected. 6 Cells, using BD Horizon TM It can be fixed with reactive staining 780, and after washing, it can be treated with TruStainFCX. TM Plus blocking. Then, staining was performed in the dark at 4°C for 30 min with a mixture of FITC anti-mouse CD11c and PE anti-mouse CD86 (BioLegend, 159203), followed by washing, resuspending, and analysis by flow cytometry. The results showed that LCAP LNP significantly upregulated CD86 expression on the surface of DCs, promoting the transformation of DCs from an immature state to an immunogenic mature phenotype. Figure 6 ).

[0057] LCAP-LNP stimulation of mouse spleen lymphocyte proliferation: Spleens were obtained from young mice. Red blood cells were lysed, and spleen cells were washed with serum-free culture medium. Cells were labeled with CFSE dye (Invitgen, 65-0850-84, final concentration 5 μM) for 10 min in the dark at 37°C. After centrifugation, cells were washed with complete culture medium to remove free CFSE. Cells were then seeded into 6-well plates and incubated at 37°C with 5% CO2. The experiment was divided into 5 groups: blank control group (0.01M PBS), blank LNP group, LCAP group, LCAP-LNP group, and positive control group (lipopolysaccharide LPS, 1 μg / mL). All treatments were adjusted to 100 μg / mL, filtered through a 0.22 μm membrane, and added at a rate of 10 μL / well. Cells were incubated for 48 h, and after treatment, cells were collected, washed twice with PBS, and counted. 2 × 10⁻⁶ cells / well 6 Cells via BD Horizon TM Immobilized cells were stained with 780 staining solution and washed before flow cytometry analysis. In contrast, the LCAP LNP group showed a significant leftward shift and multi-peak distribution of CSFE fluorescence intensity, indicating that T cells underwent multiple rounds of proliferation, suggesting that the co-delivery system can more effectively elicit an adaptive immune response. Figure 7 ).

[0058] Multiplex cytokine assay: DC2.4 cells were treated according to the LCAP LNP stimulation method for antigen presentation in DC2.4 cells. After incubation for 48 h, the culture supernatant was collected by centrifugation and analyzed using Luminex and Luminex-200 instruments. The results showed that this preparation could induce a strong pro-inflammatory microenvironment, which was beneficial to the activation of cellular immune responses. Figure 8 ).

[0059] Example 5: Humoral and cellular immune responses induced by LCAP and LNP in mice

[0060] Animal Immunization: Thirty 8-week-old SPF-grade female BALB / c mice were randomly divided into 5 groups of 6 mice each: the control group was immunized with 100 μL 0.01M PBS; the LCAP LNP group was immunized with 2.5 μg LCAP LNP; the G mRNA LNP group was immunized with 5 μg G mRNA-LNP; the G mRNA-LNP+LCAP LNP group was immunized with a physical mixture of 5 μg G mRNA LNP and 2.5 μg LCAP LNP; and the G mRNA+LCAP LNP group was immunized with LNP encapsulated from a mixture of 5 μg G mRNA and 2.5 μg LCAP. Immunization was performed via intramuscular injection in the hind leg, with a booster immunization two weeks after the initial immunization. Four weeks after the initial immunization, blood was collected via the retroorbital venous plexus, and serum was separated to detect antibody titers.

[0061] Antibody binding: To verify whether LCAP LNP could induce humoral immunity in mice, enzyme-linked immunosorbent assay (ELISA) was used to detect antigen-specific IgG titers. The results showed that the co-encapsulated G mRNA + LCAP LNP strategy was more effective in inducing high-titer antigen-specific IgG, thereby stimulating strong humoral immunity in mice. Figure 9 ).

[0062] Neutralizing Antibodies: To verify whether LCAP LNP could stimulate the production of neutralizing antibodies in mice, the level of virus-specific neutralizing antibodies in serum was measured. Serum samples were heat-inactivated at 56°C for 30 min. Three-fold diluted serum was incubated with 100 TCID50 of CVS-11 virus at 37°C for 1 h, and then the mixed serum was transferred to a confluent Vero cell monolayer and incubated in 96-well plates for 48 h. Cells were fixed with 80% acetone and FITC-labeled anti-rabies nucleoprotein antibody. Neutralizing titers were calculated as the reciprocal of the highest serum dilution, which neutralized infection at 50% wells, using the Reed-Mutch method. The results showed that the co-encapsulated G mRNA + LCAP LNP group induced mice to produce more functional neutralizing antibodies, indicating that LCAP LNP has significant synergistic potential as an adjuvant component. Figure 10 ).

[0063] Virus-specific T lymphocyte proliferation activity assay: The protective effect of mRNA-LNP vaccines depends not only on humoral immunity but also highly on cellular immunity. To verify whether LCAP LNP can stimulate cellular immunity in mice, mouse spleen cells were isolated 4 weeks post-immunization, and erythrocytes were lysed using lysis buffer. Cells (1×10⁻⁶) 6 Single-cell suspensions were seeded into 6-well plates and stimulated at 37°C for 6 h with a peptide pool (Genscript, 5 μg / well). The single-cell suspension was then mixed with TruStain FCX. TM Plus (anti-mouse CD16 / 32, BioLegend) was pre-incubated to block the Fc receptor, and then stained with fluorescently bound antibodies: APC-Cy7-anti-CD3 (100222), BV650-anti-α (100469), FITC-anti-CD8γ (162314), PE-Cy7-anti-interferon-α (505826), BV421-anti-α (506328), and BV605-anti-IL-2 (503829) antibodies (all from BioLegend), and further analyzed using FlowJo software (v10.8). The results showed that the G mRNA + LCAP LNP co-encapsulation group significantly increased IFN-γ secretion. + and TNF-α + CD4+ T cells ( Figure 11 ) and CD8 + T cells ( Figure 12 Simultaneous expression of IFN-γ + and TNF-α + Double-positive T cells ( Figure 13 ), and GC B cells ( Figure 14 ) and follicular helper T cells (Tfh) Figure 15 The ratio of LCAP to LNP indicates that LCAP and LNP can stimulate cellular immunity in mice.

[0064] Protective efficacy of the vaccine against rabies virus (RABV, CVS-24 strain) challenge: Survival rate and clinical manifestations (weight change) in a lethal challenge model are the "gold standard" indicators for evaluating the protective efficacy of rabies vaccines. To verify the protective efficacy, 8-week-old female BALB / c mice were randomly divided into 5 groups, with 5 mice in each group. Mice were immunized intramuscularly. Two weeks after the booster immunization (week 4), 100 LD50 was administered intracranially at a volume of 30 µL. 50 Mice were challenged with a dose of RABV CVS-24 strain. Mouse weight and survival were monitored daily for 14 consecutive days. The results showed that the introduction of the LCAP LNP adjuvant significantly enhanced the protective efficacy of the vaccine, providing complete protection against lethal viral attack. Figure 16 This also indicates that the vaccine effectively suppressed systemic wasting and disease progression caused by viral infection. Figure 17 ).

[0065] Example 6: Safety and toxicological evaluation of LCAP and LNP as adjuvants for vaccines

[0066] Acute toxicity in mice: Five 8-week-old SPF-grade female BALB / c mice (n=5) were intraperitoneally injected with a single high dose (containing 50 μg G mRNA and 25 μg LCAP) of G mRNA + LCAP LNP, with five mice serving as blank controls. Fourteen days post-injection, major organs (heart, liver, spleen, lung, and kidney) were collected for HE staining. Results showed that compared with the control group, the high-dose G mRNA + LCAP LNP treatment group showed no significant abnormalities in the size, color, or outline of major organs such as the heart, liver, spleen, lung, and kidney, and no pathological changes such as swelling, hemorrhage, necrosis, or significant atrophy were observed. Figure 18 No obvious inflammatory cell infiltration, tissue necrosis, or pathological damage was found in any of the sections. Figure 19 This indicates that the formulation has good tissue compatibility. Meanwhile, G mRNA+LCAPLNP did not induce a "cytokine storm" or a systemic nonspecific inflammatory response in vivo. Figure 20 ).

[0067] Obviously, the above embodiments of the present invention are merely examples to illustrate the present invention more clearly, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is impossible to exhaustively list all implementation methods here. Any obvious variations or modifications derived from the technical solutions of the present invention are still within the protection scope of the present invention.

Claims

1. A method for preparing Lysimachia christinae acidic polysaccharide lipid nanoparticles (LCAP / LNP), comprising: The acidic polysaccharide LCAP from Lysimachia christinae was dissolved in water and used as the aqueous phase. Ionizable cationic lipids, cofactor phospholipids, cholesterol, and polyethylene glycol-modified lipids were dissolved in 95% ethanol in a certain proportion to form the alcohol phase. The aqueous phase and the alcohol phase were mixed using microfluidic technology to form nanoparticles. The obtained nanoparticles were purified by ultrafiltration and the outer phase was replaced with Tris-HCl buffer to obtain Lysimachia christinae acidic polysaccharide lipid nanoparticles LCAPLNP.

2. The preparation method according to claim 1, characterized in that, The aqueous phase also contains mRNA, and the mass ratio of LCAP to mRNA is 1:

1.

3. The preparation method according to claim 1, characterized in that, The molar ratio of the ionizable cationic lipid, cofactor phospholipid, cholesterol, and polyethylene glycol ester is 50:10:38.5:1.

5.

4. The preparation method according to claim 1, characterized in that, The ultrafiltration purification was performed using a 30 kDa filter membrane at 10°C and 4500 rpm. After concentrating to one-tenth of the original volume, Tris-HCl buffer was added to bring the volume back to the original volume. This process was repeated three times.

5. A kind of Lysimachia christinae acidic polysaccharide lipid nanoparticle LCAP LNP, obtained by the preparation method according to any one of claims 1 to 4.

6. The application of the Lysimachia christinae acidic polysaccharide lipid nanoparticles (LCAP LNP) as described in claim 5 in the preparation of vaccine adjuvants.

7. A vaccine adjuvant comprising the Lysimachia christinae acidic polysaccharide lipid nanoparticles LCAP LNP as described in claim 5.

8. The vaccine adjuvant according to claim 7, characterized in that, The vaccine adjuvant also includes pharmaceutical excipients.

9. An mRNA vaccine composition comprising a viral antigen or mRNA encoding the viral antigen, and the Lysimachia christinae acidic polysaccharide lipid nanoparticles LCAP LNP as described in claim 5.

10. The mRNA vaccine composition according to claim 9, characterized in that, The vaccine in question is a rabies vaccine.

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