Spleen targeting lipid nanoparticle and application thereof

Abstract

The invention belongs to the field of biological medicines, and particularly discloses spleen targeted lipid nanoparticles and application thereof in preparation of mRNA (messenger ribonucleic acid) tumor vaccines. The spleen targeted lipid nanoparticles provided by the invention have the following advantages: (1) systemic inflammatory response (such as cytokine storm) is weakened, and a necessary immune activation function is reserved to drive antigen-specific toxic T cell response; (2) the mRNA translation efficiency is improved, antigen presentation of spleen APCs is promoted, and activation, proliferation and differentiation of tumor specific toxic T cells are enhanced; and (3) high-efficiency spleen targeted delivery is realized under a low dosage, toxic and side effects of traditional LNPs are avoided, and a safer and more efficient mRNA nano-drug design framework is provided for cancer immunotherapy and other biomedical applications.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a spleen-targeted lipid nanoparticle and an application thereof in the preparation of an mRNA tumor vaccine. Background Art

[0002] In recent years, messenger RNA (mRNA) vaccines have made breakthrough progress in responding to infectious disease challenges. Clinically, timely activation of immune responses is crucial to inhibiting the exponential growth of tumor cells and preventing the formation of an immunosuppressive tumor microenvironment. Thanks to breakthroughs in new antigen screening, delivery vectors, and adjuvant technologies, mRNA vaccines can be rapidly designed and personalized, demonstrating their transformative potential in clinical oncology. The spleen, due to its vascularized structure and high density of antigen-presenting cells (APCs), is an ideal place to initiate vaccine-induced immune responses. However, precise delivery vectors need to be developed to selectively target antigen-encoded mRNA to the spleen in order to realize this potential.

[0003] Lipid nanoparticles (LNPs) are currently the most advanced mRNA vaccine delivery vectors, as exemplified by their key role in COVID-19 and respiratory syncytial virus vaccines. However, 80%-90% of mRNA vaccine recipients experience adverse reactions, of which mild to moderate symptoms (such as pain and fever) are mostly related to inflammation. Studies have shown that the components of LNPs play a key role in activating inflammatory pathways and inducing inflammatory cytokines. These inflammation-related side effects limit the dose increase and wider biomedical applications of LNPs-based nanomedicines. In addition, traditional LNPs have limited spleen targeting and anti-tumor effects at low doses, and it is difficult to balance antigen expression, APCs activation and inflammatory homeostasis.

[0004] Currently, mRNA (mRNA-sLNPs) vaccines based on spleen-selective LNPs mainly activate splenic dendritic cells through the pro-inflammatory adjuvant effect of LNPs, thereby demonstrating their strong anti-tumor response. The pro-inflammatory characteristics of mRNA vaccines are necessary for activating antigen-specific cytotoxic T cell responses because antigen presentation under non-inflammatory conditions induces immune tolerance responses mediated by regulatory T cells (Tregs). However, the excessive inflammatory activity of LNPs may limit the translation of antigen-encoding mRNA and subsequent antigen presentation, weaken the anti-tumor cellular immune response, lead to insufficient anti-tumor cell responses, and at the same time, the risk of systemic inflammation (such as cytokine storm) also threatens its safety. Therefore, balancing innate immune activation and inflammation-mediated toxicity has become the core challenge in the synthesis of mRNA-sLNPs vaccines. Existing studies have shown that by finely regulating the lipid components and stoichiometry in mRNA-LNPs, their protein expression and inflammatory characteristics can be precisely regulated. This chemical optimization strategy, which shifts from traditional enhanced inflammatory adjuvants to molecularly engineered immunomodulation, may avoid its systemic toxicity while retaining vaccine efficacy and will be of great significance in anti-tumor treatment clinically. Summary of the Invention

[0005] The main technical problem to be solved by the present invention is to provide a spleen-targeted lipid nanoparticle, which mainly achieves the following three treatment goals: (1) retaining the necessary immune activation function to drive antigen-specific cytotoxic T cell responses while reducing systemic inflammatory responses (such as cytokine storm); (2) enhancing mRNA translation efficiency, promoting antigen presentation by splenic APCs, and strengthening the activation, proliferation, and differentiation of tumor-specific cytotoxic T cells; (3) achieving efficient spleen-targeted delivery at low doses, avoiding the toxic and side effects of traditional LNPs, and providing a safer and more efficient mRNA nanodrug design framework for cancer immunotherapy and other biomedical applications.

[0006] Secondly, the present invention provides an application of a spleen-targeted lipid nanoparticle in the preparation of an mRNA vaccine.

[0007] Thirdly, the present invention provides a spleen-targeted mRNA tumor vaccine and a preparation method thereof.

[0008] To solve the above technical problems, the present invention provides the following technical solutions:

[0009] A spleen-targeting lipid nanoparticle, the raw materials for preparing the lipid nanoparticle comprising: SM-102, cholesterol, DSPC, DMG-PEG 2000, and a long-chain saturated fatty acid; the molar percentages of SM-102, cholesterol, DSPC, DMG-PEG 2000, and the long-chain saturated fatty acid are (17.5 - 25):(3.5 - 5):(13.5 - 19):(0.5 - 0.75):(50 - 65).

[0010] As a preferred embodiment of the present invention, the molar percentages of SM-102, cholesterol, DSPC, DMG-PEG 2000, and the long-chain saturated fatty acid are (20 - 23.5):(4 - 4.7):(15.5 - 18):(0.6 - 0.7):(53 - 60).

[0011] As a preferred embodiment of the present invention, the molar percentages of SM-102, cholesterol, DSPC, DMG-PEG 2000, and the long-chain saturated fatty acid are 21.5:4.3:16.5:0.7:57.

[0012] As a preferred embodiment of the present invention, the long-chain saturated fatty acid includes, but is not limited to, one or more of palmitic acid, stearic acid, etc. Preferably, it is palmitic acid.

[0013] Use of a spleen-targeting lipid nanoparticle in the preparation of an mRNA vaccine.

[0014] As a preferred embodiment of the present invention, the mRNA vaccine is a spleen-targeting mRNA vaccine.

[0015] As a preferred embodiment of the present invention, the mRNA of the mRNA vaccine encodes a tumor antigen.

[0016] Specifically, the tumor antigens include, but are not limited to, tumor-associated antigens (TAAs), tumor-specific antigens (TSAs), virus-related antigens, etc. Among them, tumor-associated antigens are antigens that are overexpressed in tumor cells but present at low levels in normal tissues. Such antigens usually have weak immunogenicity and multiple TAAs need to be used in combination to enhance the immune response, including but not limited to carcinoembryonic antigen (CEA, for solid tumors such as colorectal cancer, NCBI accession number: NM_020219.5), melanoma-associated antigens (MAGE family such as MAGE-A3, NCBI accession number: NM_005362.4; NY-ESO-1, NCBI accession number: NM_001327.3; gp100, GenBank accession number: S73003.1; tyrosinase TYR, NCBI accession number: NM_000372.5), etc. Tumor-specific antigens are generated by gene mutations in tumor cells, have highly individualized and "non-self" characteristics, can avoid autoimmunity tolerance, and are the main targets of current personalized vaccines, including but not limited to Moderna's mRNA-4157 (mRNA encoding up to 34 new antigens), KRAS G12D in pancreatic cancer (for high-frequency mutations), etc. Virus-related antigens include but are not limited to Epstein-Barr virus latent membrane protein 2 (LMP2, GenBank accession number: M87777.1), HPV E6 / E7 proteins, etc.

[0017] As a preferred embodiment of the present invention, the mRNA of the mRNA vaccine encodes an immune-stimulating protein (such as IL-12, CD40L) or a chemokine to improve the tumor microenvironment and enhance the anti-tumor effect.

[0018] As a preferred embodiment of the present invention, the applications include, but are not limited to, one or more of the following aspects:

[0019] (1) Application in the preparation of an mRNA tumor vaccine for reducing the activation of the TLR4 / MyD88 / NF-κB pathway;

[0020] (2) Application in the preparation of an mRNA tumor vaccine for enhancing spleen targeting and delivery efficiency;

[0021] (3) Application in the preparation of an mRNA tumor vaccine for enhancing antigen presentation and immune response;

[0022] (4) Application in the preparation of an mRNA tumor vaccine for improving anti-tumor efficacy;

[0023] (5) Application in the preparation of an mRNA tumor vaccine with low toxicity and dose flexibility.

[0024] Specifically, the reduction of TLR4 / MyD88 / NF-κB pathway activation includes, but is not limited to, not over-activating the MyD88-dependent TLR4-NF-κB signal, moderately promoting DC maturation and inflammatory response, enhancing antigen processing / stability, etc.

[0025] Specifically, the improvement of spleen targeting and delivery efficiency includes, but is not limited to, increasing the mRNA translation efficiency in the spleen, enhancing the spleen expression of mRNA, etc.

[0026] Specifically, the enhancement of antigen presentation and immune response includes, but is not limited to, enhancing lysosomal escape, promoting endosomal membrane fusion, improving the cytoplasmic release efficiency of mRNA, increasing the antigen expression of DC cells, activating antigen-specific CD8 + T cells, enhancing MHC-I antigen presentation, etc.

[0027] Specifically, the improvement of anti-tumor efficacy includes, but is not limited to, reducing tumor volume, remodeling the tumor microenvironment to inhibit tumor progression and metastasis (including reducing the number and area of lung metastasis nodules), increasing T cell infiltration (including increasing the CD8 + T cell density in the tumor microenvironment and enhancing the expression of the CD69 activation marker), inducing tumor cell apoptosis, etc.

[0028] Specifically, the low toxicity and dose flexibility include, but is not limited to, increasing the survival rate of dendritic cells, reducing organ pathological damage, reducing the clinical dose, etc.

[0029] A spleen-targeted mRNA tumor vaccine, wherein the mRNA tumor vaccine comprises the spleen-targeted lipid nanoparticles and the mRNA encoding tumor antigen encapsulated therein.

[0030] As a preferred embodiment of the present invention, the tumor antigen includes, but is not limited to, tumor-associated antigen, tumor-specific antigen, virus-related antigen, etc.

[0031] As a preferred embodiment of the present invention, the size of the mRNA tumor vaccine is 40 - 200 nm.

[0032] A preparation method of a spleen-targeted mRNA tumor vaccine, comprising the following steps:

[0033] Dissolve SM-102, cholesterol, DSPC, DMG-PEG 2000 and long-chain saturated fatty acid in ethanol to obtain an organic phase;

[0034] Dissolve the mRNA encoding tumor antigen in citrate buffer to obtain an aqueous phase;

[0035] Mix the organic phase and the aqueous phase according to a volume ratio of (2 - 4):1, and purify to obtain the spleen-targeted mRNA tumor vaccine.

[0036] Advantages of the present invention:

[0037] The spleen-targeting lipid nanoparticles and mRNA vaccines based on spleen-selective LNPs provided by the present invention have the following core advantages compared with the prior art:

[0038] (1) Activation of the TLR4 / NF-κB pathway by traditional LNPs can lead to varying degrees of inflammatory responses in subjects. At the same time, excessive inflammation inhibits the mRNA translation efficiency, weakens antigen presentation, and T cell activation. However, the lipid nanoparticles provided by the present invention can significantly reduce the activation of the TLR4 / MyD88 / NF-κB pathway.

[0039] (2) The mRNA translation efficiency of conventional spleen-targeting LNPs in the spleen is limited, and they are easily non-specifically taken up by the liver. However, in the present invention, IVIS imaging shows that the fluorescence intensity of SM-102-sLNPs in the spleen is 3 times that of the original formulation MC3, and the proportion of Luc-mRNA spleen expression is increased to 90%.

[0040] (3) Traditional LNPs inhibit mRNA translation due to inflammation, resulting in insufficient antigen presentation (such as low expression of SIINFEKL-MHC complex). However, the lysosomal escape of the present invention is enhanced, lipid optimization promotes endosomal membrane fusion, the mRNA cytoplasmic release efficiency is improved (the Pearson coefficient is reduced by 40% compared with MC3), and at the same time, it promotes an increase in antigen expression in DC cells, activating more antigen-specific CD8 + T cells.

[0041] (4) The tumor suppression effect of existing vaccines is limited (such as the tumor volume in the MC3 group only decreases by 55.9%). However, the tumor volume in the treatment group after adjustment of the present invention decreases by 75.1%, the number of lung metastasis nodules decreases, and T cell infiltration increases (the density of CD8 + T cells in the tumor microenvironment increases, and the expression of the CD69 activation marker is significantly enhanced).

[0042] (5) Traditional LNPs limit the dose increase due to the risk of inflammation and rely on adjuvants to enhance immune activation. However, the present invention has low toxicity (the survival rate of DC2.4 cells > 95% at a concentration of 2000 ng / mL, and there is no pathological damage to the main organs) and dose flexibility (efficient anti-tumor effects can be achieved at a low dose (0.5 mg / kg)), providing room for clinical dose optimization. Description of the Drawings

[0043] Figure 1 Characterizations of MC3sLNPs and SM-102sLNPs in the experimental examples.

[0044] Figure 2For the in vivo translation of MC3sLNPs and SM-102sLNPs encapsulating Luc-mRNA in experimental examples.

[0045] Figure 3 For the in vivo translation of sLNPs containing palmitic acid and sLNPs containing stearic acid in experimental examples.

[0046] Figure 4 For the time-course changes of cellular uptake and lysosomal escape of MC3sLNPs and SM-102sLNPs in experimental examples.

[0047] Figure 5 For the cytotoxicity assessment of MC3sLNPs-OVA and SM-102sLNPs-OVA in experimental examples.

[0048] Figure 6 For the analysis of antigen presentation and inflammatory responses of BMDCs after treatment with MC3sLNPs-OVA and SM-102sLNPs-OVA in experimental examples.

[0049] Figure 7 For the differences in immune-induced T cell subsets and antigen-specific responses of mouse MC3sLNPs-OVA and SM-102sLNPs-OVA in experimental examples.

[0050] Figure 8 For the H&E staining of the liver, spleen, lung, kidney, and heart of mice using different nano-vaccines in experimental examples.

[0051] Figure 9 For the evaluation of tumor growth and histopathological changes in B16F10-OVA tumor-bearing mice treated with PBS, MC3sLNPs-OVA, or SM-102sLNPs-OVA in experimental examples.

[0052] Figure 10 For the analysis of immune cell infiltration in tumor tissues of mice treated with PBS, MC3sLNPs-OVA, and SM-102sLNPs-OVA in experimental examples.

[0053] Figure 11 For the CD69 expression on infiltrating T cells in tumor tissues of mice treated with PBS, MC3sLNPs-OVA, and SM-102sLNPs-OVA in experimental examples.

[0054] Figure 12 For the evaluation of the tumor metastasis inhibition effects of the PBS, empty SM-102-sLNPs, MC3sLNPs-OVA, and SM-102sLNPs-OVA treatment groups in experimental examples.

[0055] Figure 13Immunohistochemical analysis of tumor-infiltrating CD8 + and CD4 + T cells in the treatment groups of PBS, empty SM-102 sLNPs, MC3 sLNPs-OVA, and SM-102 sLNPs-OVA in the experimental examples.

[0056] Figure 14 Schematic diagram of the inflammation and cellular immune balance in cancer immunotherapy mediated by SM102 sLNPs-mRNA vaccine in the experimental examples.

[0057] To more clearly illustrate the technical solutions of the embodiments of the present invention, the attached drawings obtained in the experimental examples are briefly introduced above. It should be understood that the above-mentioned attached drawings only show some experimental examples of the present invention and should not be regarded as any limitation on the protection scope of the claims. For those of ordinary skill in the art, without creative efforts, other relevant attached drawings can also be obtained based on these attached drawings. Detailed implementation manners

[0058] The technical solutions of the present invention will be clearly and completely described below in combination with specific embodiments and experimental examples. However, those skilled in the art should understand that the embodiments are only used to illustrate the technical solutions of the present invention and should not be regarded as limiting the protection scope of the present invention. Based on the following embodiments, all other implementation manners obtained by those of ordinary skill in the art without creative efforts, such as implementation manners obtained by modification, deformation, or simple substitution, shall fall within the protection scope of the present invention.

[0059] Unless otherwise specified, the experimental methods used in the following embodiments and experimental examples are all conventional methods; the raw materials (including biological materials), reagents, culture media, instruments, etc. used, unless otherwise specified, are all commonly used in the art, publicly available, or items that can be obtained through commercial channels; the terms and abbreviations involved have their conventional meanings in the art, such as PBS buffer is phosphate buffer. Among them, SM-102 (CAS: 2089251-47-6), cholesterol (CAS: 57-88-5), DSPC (CAS: 816-94-4), DMG-PEG 2000 (CAS: 160743-62-4) are all purchased from Aveiro Shanghai Pharmaceutical Technology Co., Ltd.; long-chain unsaturated fatty acids, such as stearic acid (CAS: 57-11-4), palmitic acid are purchased from Merck Life Sciences.

[0060] Example 1

[0061] This example provides a spleen-targeted lipid nanoparticle. The raw materials for preparing the lipid nanoparticle include: SM-102, cholesterol, DSPC, DMG-PEG 2000, and stearic acid. The molar percentages of SM-102, cholesterol, DSPC, DMG-PEG 2000, and stearic acid are 21.5:4.3:16.5:0.7:57.

[0062] This example also provides an application of the spleen-targeted lipid nanoparticle in preparing an mRNA tumor vaccine. The mRNA tumor vaccine is a spleen-targeted mRNA vaccine. The application includes but is not limited to:

[0063] (1) Application in preparing an mRNA tumor vaccine that reduces the activation of the TLR4 / MyD88 / NF-κB pathway;

[0064] (2) Application in preparing an mRNA tumor vaccine that improves spleen targeting and delivery efficiency;

[0065] (3) Application in preparing an mRNA tumor vaccine that enhances antigen presentation and immune response;

[0066] (4) Application in preparing an mRNA tumor vaccine that improves anti-tumor efficacy;

[0067] (5) Application in preparing an mRNA tumor vaccine that reduces toxicity.

[0068] This example also provides a spleen-targeted mRNA tumor vaccine (i.e., sLNPs-mRNA preparation), which includes the above-mentioned spleen-targeted lipid nanoparticle and mRNA encoding a tumor antigen encapsulated therein.

[0069] This example also provides a preparation method for the spleen-targeted mRNA tumor vaccine, including the following steps:

[0070] Dissolve the lipid components (SM-102, cholesterol, DSPC, DMG-PEG 2000, and stearic acid, with a molar percentage of 21.5:4.3:16.5:0.7:57) in ethanol; dissolve the mRNA encoding the tumor antigen in citrate buffer (0.1M, pH 4.5); achieve self-assembly of the nanoparticles by rapidly mixing the organic phase and the aqueous phase (volume ratio 3:1); subsequently, perform purification by ultrafiltration-mediated buffer replacement (replacing ethanol and citrate buffer with PBS) to obtain the spleen-targeted mRNA tumor vaccine.

[0071] Example 2

[0072] This example provides a spleen-targeted lipid nanoparticle. The raw materials for preparing the lipid nanoparticle include: SM-102, cholesterol, DSPC, DMG-PEG 2000, and palmitic acid. The molar percentages of SM-102, cholesterol, DSPC, DMG-PEG 2000, and palmitic acid are 21.5:4.3:16.5:0.7:57.

[0073] This example also provides an application of the spleen-targeted lipid nanoparticle in preparing an mRNA tumor vaccine, a spleen-targeted mRNA tumor vaccine, and a preparation method thereof, which are basically the same as those in Example 1.

[0074] Example 3

[0075] This example provides a spleen-targeted lipid nanoparticle. The raw materials for preparing the lipid nanoparticle include: SM-102, cholesterol, DSPC, DMG-PEG 2000, and palmitic acid. The molar percentages of SM-102, cholesterol, DSPC, DMG-PEG 2000, and palmitic acid are 20:4:15.4:0.6:60.

[0076] This example also provides an application of the spleen-targeted lipid nanoparticle in preparing an mRNA tumor vaccine, a spleen-targeted mRNA tumor vaccine, and a preparation method thereof, which are basically the same as those in Example 1.

[0077] In other examples of the present invention, the raw materials for preparing the spleen-targeted lipid nanoparticle can take any value within a given range, which basically does not affect the physical and chemical properties and biological functions of the lipid nanoparticle, as well as the properties and efficacy of the prepared mRNA tumor vaccine.

[0078] Experimental Example

[0079] I. Experimental Method

[0080] 1. Preparation and Characterization of Lipid Nanoparticles

[0081] (1) Dissolve the lipid components (MC3 or SM-102, cholesterol, DSPC, DMG-PEG 2000, and stearic acid, with a molar percentage of 21.5:4.3:16.5:0.7:57) in ethanol, and dissolve the mRNA (OVA antigen, synthesized using E. coli Poly(A) Polymerase (CAS: M082-01B), Cap 1 Capping System (CAS: M012-01B), and T7 High Yield RNA Transcription Kit (CAS: E131-01A) from Novoprotein) in citrate buffer (0.1 M, pH 4.5). Self-assembly of the nanoparticles is achieved by rapidly mixing the organic phase with the aqueous phase (volume ratio 3:1). Subsequently, the sLNPs are purified by ultrafiltration-mediated buffer exchange (replacing ethanol and citrate buffer with PBS). The prepared nanoparticles and their Tyndall effect are as shown in Figure 1 shown.

[0082] (2) Use a Zetasizer Nano ZS90 to quantitatively analyze the polydispersity index (PDI) and ζ potential of the mRNA-loaded sLNPs. All measurements are performed in triplicate using independently prepared batches of nanoparticles to ensure data reproducibility. Finally, the ultrastructural morphology of the mRNA-loaded sLNPs is observed by transmission electron microscopy (TEM). During sample preparation, the sLNPs suspension is dropped onto a copper grid and then negatively stained with phosphotungstic acid to enhance the electron contrast. The results are as shown in Figure 1 shown.

[0083] (3) To evaluate the encapsulation efficiency of mRNA in sLNPs, free mRNA (0.2 μg) or sLNPs (containing an equal amount of mRNA) is denatured at 65 °C for 10 minutes in 2×RNA loading buffer and electrophoresed in a 0.9% formaldehyde-agarose gel containing GelRed. The mRNA size is determined using a TM millennium RNA Marker. Gel imaging is performed using a Bio-Rad ChemiDoc MP system. In addition, a NanoFCM instrument is used to characterize the particle size, encapsulation efficiency, and copy number. The results are as shown in Figure 1 shown.

[0084] 2. Bioluminescence imaging

[0085] Use The Spectrum imaging system was used to perform bioluminescence imaging on mouse organs. sLNPs containing MC3 or SM-102 and loaded with the reporter gene Luc-mRNA were injected into mice via the tail vein. Imaging was performed 6 hours after injection, and the D-luciferin substrate (150 mg / kg) was injected intraperitoneally 10 minutes before signal acquisition. After euthanasia, the bioluminescence signals of ex vivo organs (heart, liver, spleen, lung, kidney) were quantified to evaluate the in vivo expression of the nanoparticles. In addition, the in vivo expression of SM-102 sLNPs containing stearic acid or palmitic acid was compared using the same method. The results are shown in Figure 2 , 3 as follows.

[0086] 3. Evaluation of in vitro uptake, lysosomal escape and cytotoxicity of mRNA-sLNPs

[0087] (1) DC2.4 cells were seeded in 35 mm glass-bottom culture dishes at a density of 5×10 4 cells / well and allowed to adhere overnight. Subsequently, the cells were treated with sLNPs containing MC3 or SM-102 and loaded with Cy5-mRNA for 1, 3, and 5 hours respectively, and then washed with PBS. Next, the cells were incubated with 100 nM Lyso-Tracker Green staining solution in the dark at 37 °C for 1.5 hours. Finally, after the cells were washed with PBS and counterstained with Hoechst 33342 (10 ng / ml), they were observed using a confocal laser scanning microscope. The results are shown in Figure 4 as follows.

[0088] (2) To evaluate the mRNA delivery efficiency, DC2.4 cells were seeded in 24-well plates at a density of 5×10 4 cells / well and cultured until 80% confluence. The cells were treated with sLNPs containing MC3 or SM-102 and loaded with Cy5-mRNA for 1, 3, and 5 hours. After washing away the unbound nanoparticles, the cells were digested with 0.25% EDTA-trypsin, washed twice with PBS, and resuspended in FACS buffer (PBS + 2% FBS). The Cy5 fluorescence signal was detected by flow cytometry. The results are shown in Figure 4 as follows.

[0089] (3) To evaluate the cytotoxicity of sLNPs, DC2.4 cells were seeded in 96-well plates at a density of 1×10 4 cells / well and allowed to adhere overnight. After treatment with sLNPs for 24 hours, the cell viability was detected using the CCK-8 reagent. Apoptosis was evaluated synchronously: after treatment, the cells were washed twice with PBS, resuspended in Annexin V binding buffer (at a density of 1×10 6(cells / mL). Transfer 100 μL of the cell suspension to a 5 mL flow cytometry tube, add 2 μL of APC Annexin V and 1 μL of propidium iodide (PI), gently vortex, and incubate in the dark for 15 minutes. Before loading onto the instrument, supplement with binding buffer and analyze using a flow cytometer. The results are shown in Figure 5 as follows.

[0090] 4. Preparation of bone marrow-derived dendritic cells (BMDCs)

[0091] Obtain bone marrow cells from the femurs and tibias of female C57BL / 6J mice by cold PBS perfusion. After centrifugation (4 °C, 300 × g, 5 minutes), culture the cells in complete RPMI-1640 medium containing 10% FBS, 100 U / mL penicillin, 100 μg / mL streptomycin, 55 μM β-mercaptoethanol, and 20 ng / mL GM-CSF. On the 3rd day of culture, supplement with an equal volume of fresh medium. After 7 days of differentiation, collect non-adherent cells for subsequent experiments.

[0092] 5. Antigen presentation and activation effects of sLNP-OVA on BMDCs

[0093] (1) To evaluate the effects of sLNPs loaded with OVA-mRNA on antigen presentation and activation of BMDCs, treat BMDCs with PBS, MC3 sLNPs-OVA, SM-102 sLNPs-OVA, or LPS for 24 hours. After incubation, resuspend the cells in FACS buffer (PBS containing 2% FBS), and label with CD11c, CD86, CD80, MHC-II, and H-2Kb antibodies binding to SIINFEKL at 4 °C for 30 minutes. After washing twice, analyze using a BD flow cytometer. Exclude dead cells by DAPI staining. The experiment includes three independent replicates. The results are shown in Figure 6 as follows.

[0094] (2) Detect the mRNA transcription levels of IL-1β and IL-6 in BMDCs by qRT-PCR. Detect the activation status of the NF-κB signaling pathway in the whole cell lysates of BMDCs by Western blot. Incubate the protein samples with anti-TLR4, MyD88, pp65, p65, IL-1β, IL-6, or β-actin antibodies (1:1000 dilution) at 4 °C overnight, and then detect the signals using donkey anti-mouse or rabbit HRP-IgG (1:5000) secondary antibodies. Finally, develop the images using the Amersham Imager 600 system and ECL developing reagent. The results are shown in Figure 6 as follows.

[0095] 6. Analysis of OVA-specific CD8 + T cell responses in the spleen and evaluation of histopathological inflammatory responses

[0096] C57BL / 6J mice were injected with PBS, OVA mRNA-loaded MC3sLNPs or SM-102sLNPs (mRNA dose: 0.5 mg per kg body weight) via the tail vein on days 0, 5, and 10. At the experimental endpoint (day 15), spleens and peripheral blood were collected to prepare single-cell suspensions. After red blood cell lysis, splenocytes and PBMCs were labeled with CD45, CD4, CD8a, and OVA tetramer fluorescent antibodies and incubated at 4°C in the dark for 30 minutes. Viable cells were identified by DAPI exclusion. Antigen-specific CD8 + T cell populations (SIINFEKL-MHC I tetramer + / CD8 + / CD45 + ) were quantified by flow cytometry. After euthanizing C57BL / 6J mice, major organs such as the heart, liver, spleen, lungs, and kidneys were surgically removed. Tissues were immediately fixed with 4% paraformaldehyde (PFA) and paraffin-embedded 72 hours later for sectioning (5 μm thickness). Histopathological inflammatory responses were evaluated by H&E staining. The results are as Figure 7 、 8 shown.

[0097] 7. Tumor treatment experiments

[0098] (1) In the B16F10-OVA therapeutic vaccine inoculation model, 6-8-week-old C57BL / 6J mice were subcutaneously inoculated with 5×10 5 B16F10-OVA cells on the right flank. On days 8 and 13 after tumor inoculation, the mice were immunized with PBS, MC3sLNPs-OVA, or SM-102sLNPs-OVA (mRNA dose: 0.5 mg per kg body weight), respectively. Tumor growth was monitored according to the established method. Euthanasia was performed when the tumor volume reached 1500 mm 3 . The tumor volume was measured every two days with vernier calipers (volume formula: 1 / 2 × length × width 2 ), and the tumor weight was recorded at the experimental endpoint. Tumor tissues were used for H&E staining and TUNEL cell apoptosis staining. CD4 + and CD8 + T cell infiltration and the expression of CD69 on their surfaces in the tumors were analyzed by flow cytometry and immunohistochemistry. The results are as Figure 9 、 10 , 11 shown.

[0099] (2) In the B16F10-OVA lung metastasis treatment model, 6-8-week-old C57BL / 6 mice were intravenously injected with 3×10 5B16F10-OVA cells were injected intravenously with PBS, empty SM-102 sLNPs, MC3 sLNPs-OVA, or SM-102 sLNPs-OVA (mRNA dose of 0.5 mg per kilogram body weight) on days 3, 8, and 13. On day 18, the mice were euthanized, and lung metastasis was evaluated by lung weight, total metastatic area, and number of metastatic nodules. Tumor-infiltrating CD4 + and CD8 + T cells were analyzed by immunohistochemistry. The results are shown in Figure 12 and 13 .

[0100] II. Experimental Results

[0101] 1. The Physicochemical Properties and Encapsulation Efficiency of MC3 and SM-102 sLNPs are Comparable

[0102] Both MC3- and SM-102-based sLNPs-mRNA formulations exhibited a distinct Tyndall effect under laser irradiation, confirming the stable dispersion of their nanoparticles in aqueous solution ( Figure 1 a). NanoFCM analysis showed that the particle sizes of MC3 sLNPs-OVA and SM-102 sLNPs-OVA were 95.8 ± 27.0 nm and 92.3 ± 24.4 nm, respectively, with similar results ( Figure 1 b). The near-neutral zeta potential (-2 mV in PBS) indicates that moderate electrostatic repulsion may contribute to colloidal stability and avoid excessive aggregation, and its polydispersity index (PDI ~ 0.15) indicates a homogeneous nanoparticle population ( Figure 1 c). Transmission electron microscopy (TEM) imaging further verified these results, showing that both formulations were uniformly spherical, with diameters consistent with those measured by NanoFCM ( Figure 1 d). These physicochemical properties suggest that both sLNPs have high potential for efficient biological delivery, as nanoparticle size (50 - 200 nm) and surface charge are key factors in cell uptake and biodistribution.

[0103] In terms of mRNA encapsulation, agarose gel electrophoresis showed that no free mRNA bands were detected in either sLNPs-OVA (in contrast to the naked mRNA control), indicating that mRNA was almost completely encapsulated within the nanoparticles ( Figure 1 e). NanoFCM quantitative analysis showed that the encapsulation efficiencies of MC3 and SM-102 sLNPs-OVA were 90.73% and 91.97%, respectively ( Figure 1 f). Notably, the mRNA loading capacities of the two formulations were similar, with an average of 3.3 mRNA molecules carried per nanoparticle and a similar distribution range (MC3-OVA: 2.0 - 5.5; SM-102-OVA: 1.5 - 5.9)(Figure 1 g). These results indicate that both lipid formulations can efficiently encapsulate mRNA and maintain consistent loading characteristics, laying the foundation for dose uniformity in subsequent biological applications.

[0104] 2. Enhanced splenic targeting of Luc-mRNA delivery by SM-102 sLNPs compared to MC3 sLNPs

[0105] The preparation processes of MC3 and SM-102 sLNPs involve mixing lipids (MC3 / SM-102, DSPC, cholesterol, DMG-PEG, and stearic acid) and encapsulating Luc-mRNA, followed by purification and injection into mice, and organ-specific imaging is performed using the IVIS system ( Figure 2 a). Both formulations showed a significant tendency for splenic targeting, with stronger bioluminescence signals in the spleen tissue than in other major organs (liver, lung, kidney, and heart) ( Figure 2 b). The spleen signal intensity of SM-102 sLNPs was approximately three times that of MC3 sLNPs, while the fluorescence intensities of both formulations were low in the liver and lung tissues. Figure 2 c). SM-102 sLNPs had higher splenic targeting efficiency, with a higher proportion of Luc-mRNA translation in the spleen than the MC3 formulation. Figure 2 d). These results indicate that the splenic targeting ability of SM-102 nanoparticles is stronger, possibly due to the influence of ionizable lipid structural differences on organ-specific accumulation or intracellular release mechanisms.

[0106] 3. Enhanced splenic targeting of Luc-mRNA delivery by sLNPs containing palmitic acid compared to sLNPs containing stearic acid

[0107] The preparation processes of sLNPs containing stearic acid or palmitic acid involve mixing lipids (SM-102, DSPC, cholesterol, DMG-PEG, and stearic acid (SA) / palmitic acid (PA), with SA / PA accounting for 57%) and encapsulating Luc-mRNA, followed by purification and injection into mice, and organ-specific imaging is performed using the IVIS system. Both formulations showed a significant tendency for splenic targeting, with stronger bioluminescence signals in the spleen tissue than in other major organs (liver, lung, kidney, and heart) ( Figure 3 a). The spleen signal intensity of PA sLNPs was approximately twice that of SA sLNPs, while the fluorescence intensities of both formulations were low in the liver and lung tissues. Figure 3 b). PA sLNPs had higher splenic targeting efficiency, with a higher proportion of Luc-mRNA translation in the spleen than the SA formulation. Figure 3 c).

[0108] 4. Higher lysosomal escape efficiency of SM-102 sLNPs with the same cellular uptake

[0109] In DC2.4 cells, the lysosomal co-localization behavior of the two nanoparticles was similar at the 1-hour time point (Cy5-mRNA co-localized with LysoTracker signal, Figure 4 a). However, the co-localization of SM-102sLNPs with lysosomes was significantly reduced at 3 hours and 5 hours. Pearson correlation analysis quantitatively showed that the R value of SM-102sLNPs was lower ( Figure 4 b), indicating a higher lysosomal escape efficiency. In addition, the fluorescence intensity distribution showed that SM-102sLNPs (red) and lysosomal signals (green) were spatially separated after long-term incubation ( Figure 4 a).

[0110] Despite the significant difference in lysosomal escape, flow cytometry and confocal microscopy analysis showed that there was no statistical difference in the cellular uptake of the two formulations at all time points (p>0.05) ( Figure 4 c, d). This indicates that the lysosomal escape advantage of SM-102 does not stem from differences in uptake ability, but rather its physicochemical properties are more conducive to endosomal membrane disruption or regulation of intracellular transport pathways.

[0111] Cytotoxicity experiments (CCK-8 method) showed that the survival rate of DC2.4 cells did not change significantly after treatment at mRNA concentrations of 0-2000 ng / mL, and there was no difference between the two formulations ( Figure 5 a). The apoptosis rate was also similar ( Figure 5 b).

[0112] 5. SM-102sLNPs enhance antigen presentation but balance the inflammatory response by moderately activating the TLR4-NF-κB pathway

[0113] Compared with MC3sLNPs-OVA, SM-102sLNPs-OVA significantly enhanced SIINFEKL antigen presentation ( Figure 6 a, e), but did not overly induce the expression of co-stimulatory molecules CD80, CD86, and MHC-II ( Figure 6 b-d, f-h). This dissociation of antigen presentation and DC activation was accompanied by moderate activation of inflammatory signals: the mRNA levels of IL-1β and IL-6 were lower than those in the MC3 group ( Figure 6 i-j), and the activity of the TLR4-NF-κB pathway was lower than that in the MC3 group (the levels of TLR4, MyD88, p-p65, and cytokine proteins were lower than those in the MC3 group, Figure 6 k-q). It shows that SM-102 can enhance antigen processing / stability while moderately promoting DC maturation and inflammatory response without overly activating the MyD88-dependent TLR4-NF-κB signal. This functional difference makes SM-102sLNPs more advantageous in controlled immunological applications.

[0114] 6. SM-102 sLNPs-OVA Elicits Stronger Systemic and Antigen-Specific T Cell Responses

[0115] After immunizing mice according to Figure 6 protocol a (dose 0.5 mg / kg), the proportions of CD4 + and CD8 + T cells in the peripheral blood and spleen of the SM-102 sLNPs-OVA group were higher than those in the MC3 group ( Figure 7 b, c). OVA tetramer staining showed that the proportions of antigen-specific CD8 + T cells in the spleen and blood of the SM-102 group were higher ( Figure 7 d, e), suggesting that it may enhance MHC-I antigen presentation or cytotoxic T cell cross-activation. H&E staining showed no significant pathological damage in each organ ( Figure 8 ), indicating good safety.

[0116] 7. SM-102 sLNPs-OVA Inhibits Tumor Growth and Metastasis by Enhancing T Cell Infiltration and Inducing Apoptosis

[0117] In B16F10-OVA tumor-bearing mice ( Figure 9 a), the tumor volume ( Figure 9 b-d) and weight ( Figure 9 e) of the SM-102 group were significantly lower than those of the PBS and MC3 groups, and there was no difference in body weight ( Figure 9 f). H&E and TUNEL staining of tumor tissues showed a decrease in cell density and an increase in apoptosis in the SM-102 group ( Figure 9 g, h). Flow cytometry and immunohistochemistry confirmed that the infiltration of CD45 + leukocytes, CD8a + and CD4 + T cells in the tumor microenvironment of the SM-102 group increased significantly ( Figure 10 a-e), and the expression of the T cell activation marker CD69 increased ( Figure 11 a, b).

[0118] In the lung metastasis model ( Figure 12 a), dense black tumor nodules were visible in the lungs of the PBS group, while the number and area of nodules in the SM-102 group were significantly reduced ( Figure 12 b-f). Immunohistochemistry showed enhanced T cell infiltration in the lung tissue of the SM-102 group ( Figure 13 a, b). In summary, SM-102 sLNPs-OVA inhibits tumor progression and metastasis by remodeling the tumor microenvironment, and the effect is better than that of the MC3 preparation.

[0119] In summary, the present invention realizes the molecular mechanism of "low inflammation - high immune activation" ( Figure 14 ), such as the association of specific signal pathway inhibition / activation. This lipid nanoparticle has a specific mode of action in enhancing antigen presentation and T cell activation in splenic APCs, and is applicable to cancer immunotherapy (such as activating tumor - specific cytotoxic T cells and inhibiting immune escape in the tumor microenvironment). In short, the key points of the technical solution of the present invention include:

[0120] (1) Molecular engineering optimization of ionizable lipids: By adjusting the lipid type and dosage, precisely regulate the inflammatory activity of lipid nanoparticles (LNPs), that is, weaken the systemic inflammatory response (such as cytokine storm), while retaining the necessary innate immune activation function to drive antigen - specific cytotoxic T cell responses.

[0121] (2) Innovative design of spleen - targeted delivery system: Improve the translation efficiency of mRNA in the spleen at low doses, enhance the antigen - presenting ability in splenic APCs, and activate tumor - specific cytotoxic T cells.

[0122] (3) Balance between immune activation and inflammatory homeostasis: By optimizing the inflammatory characteristics of LNPs, solve the contradiction of "excessive inflammation inhibition effect" in traditional LNPs.

[0123] (4) Co - enhance efficacy and safety: Achieve efficient anti - tumor immune responses (such as T cell activation and proliferation) at low doses, and avoid the side effects of traditional LNPs. It is applicable to cancer immunotherapy and provides a new paradigm for the development of other mRNA nanomedicines.

[0124] Although the technical solutions of the present invention have been described in detail above with general descriptions, specific embodiments and experimental examples, it should be noted that the embodiments and experimental examples are only used to illustrate the technical solutions and technical effects of the present invention, and should not be regarded as any limitation to the protection scope of the present invention. Simple deformations, modifications or improvements based on the technical concept of the present invention all fall within the scope of protection required by the present invention.

Claims

1. A spleen-targeting lipid nanoparticle, characterized in that: The raw materials for preparing the lipid nanoparticles include: SM-102, cholesterol, DSPC, DMG-PEG 2000, and long-chain saturated fatty acids; the molar percentages of SM-102, cholesterol, DSPC, DMG-PEG 2000, and long-chain saturated fatty acids are (17.5 - 25):(3.5 - 5):(13.5 - 19):(0.5 - 0.75):(50 - 65).

2. The lipid nanoparticle according to claim 1, wherein: The molar percentages of SM-102, cholesterol, DSPC, DMG-PEG 2000, and long-chain saturated fatty acids are (20 - 23.5):(4 - 4.7):(15.5 - 18):(0.6 - 0.7):(53 - 60).

3. The lipid nanoparticle according to claim 1, wherein: The molar percentages of SM-102, cholesterol, DSPC, DMG-PEG 2000, and long-chain saturated fatty acids are 21.5:4.3:16.5:0.7:57; And / or, the long-chain saturated fatty acids include, but are not limited to, one or more of palmitic acid and stearic acid.

4. Use of a spleen-targeting lipid nanoparticle as described in any one of claims 1 - 3 in the preparation of an mRNA vaccine.

5. The application according to claim 4, characterized in that: The mRNA vaccine is a spleen-targeting mRNA vaccine; And / or, the mRNA of the mRNA vaccine encodes a tumor antigen; And / or, the mRNA of the mRNA vaccine encodes an immune-stimulating protein or chemokine.

6. The application according to claim 4, characterized in that: The use includes, but is not limited to, one or more of the following aspects: (1) Use in the preparation of an mRNA tumor vaccine for reducing the activation of the TLR4 / MyD88 / NF-κB pathway; (2) Use in the preparation of an mRNA tumor vaccine for enhancing spleen targeting and delivery efficiency; (3) Use in the preparation of an mRNA tumor vaccine for enhancing antigen presentation and immune response; (4) Use in the preparation of an mRNA tumor vaccine for improving anti-tumor efficacy; (5) Use in the preparation of an mRNA tumor vaccine with low toxicity and dose flexibility.

7. The application according to claim 6, characterized in that: The reduction of TLR4 / MyD88 / NF-κB pathway activation includes, but is not limited to, not over-activating the MyD88-dependent TLR4-NF-κB signal, moderately promoting DC maturation and inflammatory response, and enhancing antigen processing / stability; And / or, the enhancement of spleen targeting and delivery efficiency includes, but is not limited to, increasing the mRNA translation efficiency in the spleen and increasing the spleen expression of mRNA; And / or, the enhancement of antigen presentation and immune response includes but is not limited to enhancing lysosomal escape, promoting endosomal membrane fusion, improving the efficiency of mRNA cytoplasmic release, increasing antigen expression in DC cells, activating antigen-specific CD8 + T cells, enhancing MHC-I antigen presentation; And / or, the improvement of anti-tumor efficacy includes, but is not limited to, reducing tumor volume, inhibiting tumor progression and metastasis, increasing T cell infiltration, and inducing tumor cell apoptosis; And / or, the low toxicity and dose flexibility includes, but is not limited to, increasing dendritic cell survival rate, reducing organ pathological damage, and reducing clinical dose.

8. A spleen-targeted mRNA tumor vaccine, characterized in that: The mRNA tumor vaccine includes a spleen-targeting lipid nanoparticle as described in any one of claims 1 - 3 and mRNA encoding a tumor antigen encapsulated therein.

9. The mRNA tumor vaccine according to claim 8, wherein: The tumor antigen includes, but is not limited to, tumor-associated antigen, tumor-specific antigen, and virus-related antigen; And / or, the size of the mRNA tumor vaccine is 40 - 200 nm.

10. A method for preparing a spleen-targeted mRNA tumor vaccine according to any one of claims 8-9, characterized in that: The preparation method includes the following steps: Dissolve SM-102, cholesterol, DSPC, DMG-PEG 2000 and long-chain saturated fatty acid in ethanol to obtain an organic phase; Dissolve the mRNA encoding tumor antigen in citrate buffer to obtain an aqueous phase; Mix the organic phase and the aqueous phase at a volume ratio of (2-4):1, and after purification, obtain a spleen-targeted mRNA tumor vaccine.

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