Spleen-targeting lipid nanoparticles and uses thereof

Abstract

The application belongs to the field of biological medicine, and specifically discloses a spleen-targeting lipid nanoparticle and application thereof in preparation of an mRNA tumor vaccine. The spleen-targeting lipid nanoparticle provided by the application has the following advantages: (1) weakening of systemic inflammatory response (such as cytokine storm) while retaining necessary immune activation function to drive antigen-specific toxic T cell response; (2) improvement of mRNA translation efficiency, promotion of antigen presentation of spleen APCs, and strengthening of activation, proliferation and differentiation of tumor-specific toxic T cells; and (3) realization of efficient spleen-targeting delivery at a low dose, avoidance of toxic side effects of traditional LNPs, and provision of a safer and more efficient mRNA nanoparticle drug design framework for cancer immunotherapy and other biomedical applications.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a spleen-targeting lipid nanoparticle and its application in the preparation of mRNA tumor vaccines. Background Technology

[0002] In recent years, messenger RNA (mRNA) vaccines have made groundbreaking progress in addressing the challenges of infectious diseases. Clinically, timely activation of the immune response is crucial for inhibiting the exponential growth of tumor cells and preventing the formation of an immunosuppressive tumor microenvironment. Thanks to breakthroughs in neoantigen 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 site for initiating vaccine-induced immune responses. However, this potential needs to be realized by developing precise delivery vectors to selectively target and deliver antigen-encoded mRNA to the spleen.

[0003] Lipid nanoparticles (LNPs) are currently the most advanced mRNA vaccine delivery vectors, as evidenced by their crucial role in COVID-19 and respiratory syncytial virus (RSV) vaccines. However, 80%-90% of mRNA vaccine recipients experience adverse reactions, with mild to moderate symptoms (such as pain and fever) often related to inflammation. Studies have shown that components of LNPs play a key role in activating inflammatory pathways and inducing inflammatory cytokines. These inflammation-related side effects limit the dosage increase and broader biomedical applications of LNP-based nanomedicines. Furthermore, traditional LNPs exhibit limited spleen targeting and antitumor efficacy at low doses, making it difficult to balance antigen expression, APC activation, and inflammatory homeostasis.

[0004] Currently, mRNA-sLNPs-based vaccines primarily exhibit potent anti-tumor responses by activating splenic dendritic cells through the pro-inflammatory adjuvant effect of LNPs. The pro-inflammatory properties of mRNA vaccines are essential for activating antigen-specific toxic T cell responses, as antigen presentation under non-inflammatory conditions induces regulatory T cell (Treg)-mediated immune tolerance. However, excessive inflammatory activity of LNPs may limit the translation of antigen-encoded mRNA and subsequent antigen presentation, weakening anti-tumor cellular immune responses and leading to insufficient anti-tumor cellular responses. Furthermore, systemic inflammatory risks (such as cytokine storms) threaten their safety. Therefore, balancing innate immune activation with inflammation-mediated toxicity is a core challenge in the synthesis of mRNA-sLNPs vaccines. Previous studies have shown that by finely regulating the lipid components and stoichiometry in mRNA-LNPs, their protein expression and inflammatory characteristics can be precisely modulated. This chemical optimization strategy, shifting from traditional adjuvant enhancement to molecularly engineered immunomodulation, may preserve vaccine efficacy while mitigating systemic toxicity, holding significant implications for clinical anti-tumor therapy. Summary of the Invention

[0005] The main technical problem solved by this invention is to provide a spleen-targeting lipid nanoparticle that achieves the following three therapeutic goals: (1) while reducing systemic inflammatory responses (such as cytokine storms), it retains the necessary immune activation function to drive antigen-specific toxic T cell responses; (2) it improves mRNA translation efficiency, promotes antigen presentation of spleen APCs, and enhances the activation, proliferation and differentiation of tumor-specific toxic T cells; (3) it achieves efficient spleen-targeted delivery at low doses, avoids the toxic side effects of traditional LNPs, and provides a safer and more efficient mRNA nanomedicine design framework for cancer immunotherapy and other biomedical applications.

[0006] Secondly, this invention provides the application of spleen-targeting lipid nanoparticles in the preparation of mRNA vaccines.

[0007] Furthermore, this invention provides a spleen-targeting mRNA tumor vaccine and its preparation method.

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

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

[0010] In a preferred embodiment of the present invention, 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).

[0011] In a preferred embodiment of the present invention, 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.

[0012] In 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. Palmitic acid is preferred.

[0013] Application of spleen-targeting lipid nanoparticles in the preparation of mRNA vaccines.

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

[0015] In 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), and virus-associated antigens. Tumor-associated antigens are antigens that are overexpressed in tumor cells but present at low levels in normal tissues. These antigens typically have weak immunogenicity and require the combined use of multiple TAAs to enhance the immune response. These include, but are not limited to, carcinoembryonic antigen (CEA, for solid tumors such as colorectal cancer, NCBI accession number: NM_020219.5) and 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). Tumor-specific antigens (TBA) are generated by gene mutations in tumor cells. They are highly individualized and "non-self" in nature, avoiding autoimmune tolerance, and are the main targets of current personalized vaccines. Examples include, but are not limited to, Moderna's mRNA-4157 (encoding up to 34 neoantigens) and KRAS G12D in pancreatic cancer (targeting high-frequency mutations). Virus-associated antigens (VAA) include, but are not limited to, EBV latent membrane protein 2 (LMP2, GenBank accession number: M87777.1) and HPV E6 / E7 proteins.

[0017] In a preferred embodiment of the present invention, the mRNA vaccine encodes immunostimulatory proteins (such as IL-12, CD40L) or chemokines to improve the tumor microenvironment and enhance the anti-tumor effect.

[0018] As a preferred embodiment of the present invention, the application includes, but is not limited to, one or more of the following aspects:

[0019] (1) Application in the preparation of mRNA tumor vaccines that reduce the activation of the TLR4 / MyD88 / NF-κB pathway;

[0020] (2) Application in the preparation of mRNA tumor vaccines that enhance spleen targeting and delivery efficiency;

[0021] (3) Application in the preparation of mRNA tumor vaccines that enhance antigen presentation and immune response;

[0022] (4) Application in the preparation of mRNA tumor vaccines that enhance anti-tumor efficacy;

[0023] (5) Application in the preparation of mRNA tumor vaccines with low toxicity and dosage flexibility.

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

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

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

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

[0028] Specifically, the low toxicity and dosage flexibility include, but are not limited to, improving dendritic cell survival, reducing organ pathological damage, and reducing clinical dosage.

[0029] A spleen-targeting mRNA tumor vaccine, the mRNA tumor vaccine comprising spleen-targeting lipid nanoparticles and mRNA encoding a tumor antigen encapsulated therein.

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

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

[0032] A method for preparing a spleen-targeting mRNA tumor vaccine includes the following steps:

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

[0034] The mRNA encoding tumor antigen was dissolved in citrate buffer to obtain an aqueous solution;

[0035] The organic phase and the aqueous phase were mixed at a volume ratio of (2-4):1, and the resulting purified mixture was used to obtain a spleen-targeted mRNA tumor vaccine.

[0036] The beneficial effects of this invention are:

[0037] The spleen-targeting lipid nanoparticles and spleen-selective LNP-based mRNA vaccines provided by this invention have the following core advantages compared to existing technologies:

[0038] (1) Traditional LNPs activate the TLR4 / NF-κB pathway, leading to varying degrees of inflammatory response in subjects. Excessive inflammation also inhibits mRNA translation efficiency, weakening antigen presentation and T cell activation. However, the lipid nanoparticles provided in this invention can significantly reduce the activation of the TLR4 / MyD88 / NF-κB pathway.

[0039] (2) Conventional spleen-targeting LNPs have limited mRNA translation efficiency in the spleen and are easily taken up non-specifically by the liver. However, in this invention, IVIS imaging showed that the fluorescence intensity of SM-102-sLNPs in the spleen was 3 times that of the original formulation MC3, and the Luc-mRNA expression ratio in the spleen was increased to 90%.

[0040] (3) Traditional LNPs suffer from insufficient antigen presentation due to inflammation-induced suppression of mRNA translation (e.g., low expression of the SIINFEKL-MHC complex). In contrast, the present invention enhances lysosomal escape, optimizes lipids to promote endosome membrane fusion, and improves mRNA cytoplasmic release efficiency (Pearson coefficient reduced by 40% compared to MC3). Simultaneously, it increases antigen expression in DC cells and activates more antigen-specific CD8+ cells. + T cells.

[0041] (4) Existing vaccines have limited tumor-suppressing effects (e.g., tumor volume in the MC3 group decreased by only 55.9%). In contrast, the adjusted treatment group of this invention experienced a 75.1% reduction in tumor volume, a decrease in the number of lung metastatic nodules, and an increase in T-cell infiltration (CD8+ in the tumor microenvironment). + T cell density increased, and expression of CD69 activation markers was significantly enhanced.

[0042] (5) Traditional LNPs have limited dosage increases due to inflammatory risks and require adjuvants to enhance immune activation. In contrast, this invention has low toxicity (DC2.4 cell survival rate >95% at a concentration of 2000 ng / mL, with no pathological damage to major organs) and dosage flexibility (highly effective anti-tumor effect can be achieved at a low dose (0.5 mg / kg), providing room for clinical dosage optimization. Attached Figure Description

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

[0044] Figure 2This study describes the in vivo translation of MC3sLNPs and SM-102sLNPs that encapsulate Luc-mRNA in the experimental example.

[0045] Figure 3 This refers to the in vivo translation of palmitic acid-containing sLNPs and stearic acid-containing sLNPs in the experimental examples.

[0046] Figure 4 The time-dynamic changes in cellular uptake and lysosomal escape of MC3sLNPs and SM-102sLNPs in the experimental case are shown.

[0047] Figure 5 The cytotoxicity of MC3sLNPs-OVA and SM-102sLNPs-OVA in the experimental cases was evaluated.

[0048] Figure 6 This study analyzed antigen presentation and inflammatory response of BMDCs after treatment with MC3sLNPs-OVA and SM-102sLNPs-OVA in the experimental case.

[0049] Figure 7 The differences in immune-induced T cell subsets and antigen-specific responses between MC3sLNPs-OVA and SM-102sLNPs-OVA in the experimental mice were observed.

[0050] Figure 8 H&E staining was performed on the liver, spleen, lungs, kidneys, and heart of mice using different nanovaccines in the experimental case.

[0051] Figure 9 To evaluate tumor growth and histopathological changes in B16F10-OVA-treated mice with PBS, MC3sLNPs-OVA or SM-102sLNPs-OVA in experimental cases.

[0052] Figure 10 This study analyzed the immune cell infiltration in mouse tumor tissues treated with PBS, MC3sLNPs-OVA, and SM-102sLNPs-OVA in the experimental cases.

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

[0054] Figure 12 The study aimed to evaluate the tumor metastasis inhibition effects of PBS, empty SM-102-sLNPs, MC3sLNPs-OVA, and SM-102sLNPs-OVA treatment groups in the experimental cases.

[0055] Figure 13The experimental group included PBS, empty vector SM-102sLNPs, MC3sLNPs-OVA, and SM-102sLNPs-OVA treatment groups, which showed tumor-infiltrating CD8+. + and CD4 + Immunohistochemical analysis of T cells.

[0056] Figure 14 This is a schematic diagram illustrating the inflammation and cellular immune balance in cancer immunotherapy mediated by the SM102sLNPs-mRNA vaccine in the experimental case.

[0057] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings obtained in the experimental examples have been briefly described above. It should be understood that the above drawings only show some experimental examples of the present invention and should not be considered as any limitation on the scope of protection of the claims. For those skilled in the art, other related drawings can be obtained based on these drawings without any creative effort. Detailed Implementation

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

[0059] Unless otherwise specified, the experimental methods used in the following examples and experimental cases are conventional methods; the raw materials (including biological materials), reagents, culture media, instruments, etc., used are all commonly used in the field and are publicly available or commercially available unless otherwise specified; the terms and abbreviations used have their conventional meanings in the field, such as PBS buffer being phosphate buffer. SM-102 (CAS: 2089251-47-6), cholesterol (CAS: 57-88-5), DSPC (CAS: 816-94-4), and DMG-PEG 2000 (CAS: 160743-62-4) were all purchased from Aivit Shanghai Pharmaceuticals Technology Co., Ltd.; long-chain unsaturated fatty acids, such as stearic acid (CAS: 57-11-4) and palmitic acid, were purchased from Merck Life Sciences.

[0060] Example 1

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

[0062] This embodiment also provides an application of spleen-targeting lipid nanoparticles in the preparation of mRNA tumor vaccines; the mRNA tumor vaccine is a spleen-targeting mRNA vaccine; the application includes, but is not limited to:

[0063] (1) Application in the preparation of mRNA tumor vaccines that reduce the activation of the TLR4 / MyD88 / NF-κB pathway;

[0064] (2) Application in the preparation of mRNA tumor vaccines that improve spleen targeting and delivery efficiency;

[0065] (3) Application in the preparation of mRNA tumor vaccines that enhance antigen presentation and immune response;

[0066] (4) Application in the preparation of mRNA tumor vaccines that enhance anti-tumor efficacy;

[0067] (5) Application in the preparation of mRNA tumor vaccines with reduced toxicity.

[0068] This embodiment also provides a spleen-targeting mRNA tumor vaccine (i.e., sLNPs-mRNA formulation), comprising the above-mentioned spleen-targeting lipid nanoparticles and mRNA encoding tumor antigens encapsulated therein.

[0069] This embodiment also provides a method for preparing a spleen-targeting mRNA tumor vaccine, including the following steps:

[0070] The lipid components (SM-102, cholesterol, DSPC, DMG-PEG 2000, and stearic acid, in molar percentages of 21.5:4.3:16.5:0.7:57) were dissolved in ethanol; the mRNA encoding the tumor antigen was dissolved in citrate buffer (0.1M, pH 4.5); the self-assembly of nanoparticles was achieved by rapidly mixing the organic and aqueous phases (volume ratio 3:1); and the spleen-targeting mRNA tumor vaccine was obtained by ultrafiltration-mediated buffer replacement (replacing ethanol and citrate buffer with PBS).

[0071] Example 2

[0072] This embodiment provides a spleen-targeting 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 embodiment also provides the application of spleen-targeting lipid nanoparticles in the preparation of mRNA tumor vaccines, spleen-targeting mRNA tumor vaccines and their preparation methods, which are basically the same as in Embodiment 1.

[0074] Example 3

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

[0076] This embodiment also provides the application of spleen-targeting lipid nanoparticles in the preparation of mRNA tumor vaccines, spleen-targeting mRNA tumor vaccines and their preparation methods, which are basically the same as in Embodiment 1.

[0077] In other embodiments of the present invention, the raw materials for preparing spleen-targeting lipid nanoparticles can be arbitrarily selected within a given range without significantly affecting the physicochemical properties and biological functions of the lipid nanoparticles, as well as the properties and efficacy of the prepared mRNA tumor vaccine.

[0078] Experimental Example

[0079] I. Experimental Methods

[0080] 1. Preparation and characterization of lipid nanoparticles

[0081] (1) 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) were dissolved in ethanol, and 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)) was dissolved in citrate buffer (0.1M, pH 4.5). Nanoparticle self-assembly was achieved by rapidly mixing the organic and aqueous phases (volume ratio 3:1). The sLNPs were then purified by ultrafiltration-mediated buffer replacement (replacing ethanol and citrate buffer with PBS). The prepared nanoparticles and their Tyndall effect were as follows: Figure 1 As shown.

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

[0083] (3) To assess the encapsulation efficiency of mRNA in sLNPs, free mRNA (0.2 μg) or sLNPs (containing an equal amount of mRNA) were denatured in 2×RNA loading buffer at 65°C for 10 min and electrophoresed on a 0.9% formaldehyde-agarose gel containing GelRed. mRNA size was determined by millennium... TM RNA markers were identified. Gel imaging was performed using the Bio-Rad ChemiDoc MP system. Furthermore, particle size, encapsulation efficiency, and copy number were characterized using a NanoFCM instrument. Results are as follows: Figure 1 As shown.

[0084] 2. Bioluminescence imaging

[0085] use Bioluminescence imaging of mouse organs was performed using the Spectrum imaging system. Mouse sLNPs carrying the reporter gene Luc-mRNA and containing either MC3 or SM-102 were injected via tail vein injection. Imaging was performed 6 hours post-injection, with D-luciferin substrate (150 mg / kg) administered intraperitoneally 10 minutes before signal acquisition. After euthanasia, bioluminescence signals from ex vivo organs (heart, liver, spleen, lung, and kidney) were quantified to assess in vivo nanoparticle expression. Furthermore, the in vivo expression of SM-102 sLNPs containing stearic acid or palmitic acid was compared using the same method. Results are as follows: Figure 2 , 3 As shown.

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

[0087] (1) DC2.4 cells were inoculated at 5 × 10⁻⁶ cells per cell line. 4 Cells were seeded at a density of [number] cells / well in 35 mm glass-bottomed culture dishes and allowed to adhere overnight. Cells were then treated with sLNPs containing MC3 or SM-102 loaded with Cy5-mRNA for 1, 3, and 5 hours, respectively, and washed with PBS. Next, cells were incubated with 100 nM Lyso-Tracker Green staining solution at 37°C in the dark for 1.5 hours. Finally, after washing with PBS and counterstaining with Hoechst 33342 (10 ng / ml), cells were observed using a confocal laser scanning microscope. Results are as follows: Figure 4 As shown.

[0088] (2) To evaluate mRNA delivery efficiency, DC2.4 cells were subjected to a 5 × 10⁻⁶ mRNA delivery rate. 4 Cells were seeded at a density of [number] cells / well in 24-well plates and cultured to 80% confluence. Cells were treated with sLNPs containing MC3 or SM-102 loaded with Cy5-mRNA for 1, 3, and 5 hours. After washing away unbound nanoparticles, cells were digested with 0.25% EDTA-trypsin, washed twice with PBS, and resuspended in FACS buffer (PBS + 2% FBS). Cy5 fluorescence signal was detected by flow cytometry. Results are shown below. Figure 4 As shown.

[0089] (3) To assess the cytotoxicity of sLNPs, DC2.4 cells were sputtered at a concentration of 1×10⁻⁶. 4 Cells were seeded at a density of 1 cell / well in 96-well plates and incubated overnight. Cell viability was assessed using CCK-8 assay 24 hours after sLNP treatment. Apoptosis was assessed concurrently: cells were washed twice with PBS after treatment and resuspended in Annexin V binding buffer (density 1×10⁻⁶). 6(cells / mL). Transfer 100 μL of 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. Add binding buffer before flow cytometry analysis. Results are as follows: Figure 5 As shown.

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

[0091] Bone marrow cells were obtained from the femur and tibia of female C57BL / 6J mice via cold PBS perfusion. After centrifugation (4°C, 300×g, 5 min), cells were cultured 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. An equal volume of fresh medium was added on day 3 of culture. After 7 days of differentiation, non-adherent cells were collected for subsequent experiments.

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

[0093] (1) To evaluate the effect of OVA-mRNA-loaded sLNPs on antigen presentation and activation of BMDCs, BMDCs were treated with PBS, MC3sLNPs-OVA, SM-102sLNPs-OVA, or LPS for 24 hours. After incubation, cells were resuspended in FACS buffer (PBS containing 2% FBS) and labeled with CD11c, CD86, CD80, MHC-II, and H-2Kb antibody bound to SIINFEKL at 4°C for 30 minutes. After washing twice, cells were analyzed by BD flow cytometry. Dead cells were excluded by DAPI staining. The experiment consisted of three independent replicates. Results are as follows: Figure 6 As shown.

[0094] (2) The mRNA transcription levels of IL-1β and IL-6 in BMDCs were detected by qRT-PCR. The activation status of the NF-κB signaling pathway in whole-cell lysates of BMDCs was detected by Western blot. Protein samples were incubated overnight at 4°C with antibodies against TLR4, MyD88, pp65, p65, IL-1β, IL-6, or β-actin (1:1000 dilution), followed by signal detection using donkey anti-mouse or rabbit HRP-IgG (1:5000) secondary antibody. Finally, the samples were developed using an Amersham Imager 600 system and ECL imaging reagent. Results are as follows: Figure 6 As shown.

[0095] 6. OVA-specific CD8 in the spleen + T-cell response analysis and histopathological assessment of inflammatory response

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

[0097] 7. Tumor Treatment Experiments

[0098] (1) In the B16F10-OVA therapeutic vaccination model, 6-8 week old C57BL / 6J mice were subcutaneously injected with 5×10 oz. 5 B16F10-OVA cells were inoculated. On days 8 and 13 post-inoculation, tumor cells were immunized with PBS, MC3sLNPs-OVA, or SM-102sLNPs-OVA (mRNA dose of 0.5 mg / kg body weight), respectively. Tumor growth was monitored according to established methods. When the tumor volume reached 1500 mm², the tumor was immunized. 3 Euthanasia was performed at that time. The tumor volume was measured every two days using calipers (volume formula: 1 / 2×length×width 2 Tumor weight was recorded as the experimental endpoint. Tumor tissue was used for H&E staining and TUNEL cell apoptosis staining. CD4+ within the tumor was analyzed by flow cytometry and immunohistochemistry. + and CD8 + T cell infiltration and the expression of CD69 on its surface. Results are as follows: Figure 9 , 10 As shown in Figure 11.

[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 intravenously injected with PBS, empty vector SM-102sLNPs, MC3sLNPs-OVA, or SM-102sLNPs-OVA (mRNA dose of 0.5 mg / kg body weight) on days 3, 8, and 13. Cells were euthanized on day 18, and lung metastasis was assessed by lung weight, total metastatic area, and number of metastatic nodules. Immunohistochemical analysis of intratumoral CD4+ was performed. + and CD8 + T cell infiltration. Results as follows: Figure 12 , 13 As shown.

[0100] II. Experimental Results

[0101] 1. The physicochemical properties and encapsulation efficiency of MC3 and SM-102sLNPs are comparable.

[0102] Both sLNPs-mRNA formulations based on MC3 and SM-102 exhibited a significant 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 MC3sLNPs-OVA and SM-102sLNPs-OVA were 95.8±27.0 nm and 92.3±24.4 nm, respectively, which were similar. Figure 1 b). The near-neutral zeta potential (-2 mV in PBS) suggests that moderate electrostatic repulsion may contribute to colloidal stability and prevent excessive aggregation, and its polydispersity index (PDI ~ 0.15) indicates that the nanoparticle population is homogeneous. Figure 1 c). Transmission electron microscopy (TEM) imaging further validated these results, showing that both formulations were uniformly spherical with diameters consistent with NanoFCM measurements. Figure 1 d). These physicochemical properties indicate that both sLNPs have the potential for efficient biodelivery, as the size (50-200 nm) and surface charge of nanoparticles are key factors for cellular uptake and biodistribution.

[0103] Regarding 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 the mRNA was almost completely encapsulated within the nanoparticles. Figure 1 e). NanoFCM quantitative analysis showed that the encapsulation efficiencies of MC3 and SM-102sLNPs-OVA were 90.73% and 91.97%, respectively. Figure 1 f). Notably, the two formulations have similar mRNA loading capacities, with an average of 3.3 mRNA molecules carried per nanoparticle, and similar distribution ranges (MC3-OVA: 2.0-5.5; SM-102-OVA: 1.5-5.9). Figure 1 These results demonstrate that both lipid formulations efficiently encapsulate mRNA while maintaining consistent loading characteristics, laying the foundation for dose uniformity in subsequent biological applications.

[0104] 2. SM-102sLNPs enhance spleen-targeted Luc-mRNA delivery compared to MC3sLNPs.

[0105] The preparation process of MC3 and SM-102sLNPs involves mixing lipids (MC3 / SM-102, DSPC, cholesterol, DMG-PEG, and stearic acid) and encapsulating Luc-mRNA, followed by purification and injection into mice for organ-specific imaging using an IVIS system. Figure 2 a) Both formulations showed a significant spleen-targeting tendency, with stronger bioluminescent signals in spleen tissue compared to other major organs (liver, lung, kidney, and heart). Figure 2 b). The spleen signal intensity of SM-102sLNPs was approximately three times that of MC3sLNPs, while both formulations showed low fluorescence intensity in liver and lung tissues. Figure 2 c). SM-102sLNPs exhibit higher spleen-targeting efficiency, with a higher proportion of Luc-mRNA translation in the spleen compared to MC3 formulations. Figure 2 d). These results suggest that the spleen-targeting ability based on SM-102 nanoparticles is stronger, which may be due to the influence of differences in ionizable lipid structure on organ-specific accumulation or intracellular release mechanisms.

[0106] 3. Palmitic acid-containing sLNPs enhance spleen-targeted Luc-mRNA delivery compared to stearic acid-containing sLNPs.

[0107] The preparation of sLNPs containing stearic acid or palmitic acid involves 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 using an IVIS system. Both formulations showed a significant spleen-targeting tendency, with stronger bioluminescent signals in spleen tissue compared to 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 intensity of both formulations was low in liver and lung tissue. Figure 3 b). PAsLNPs have higher spleen-targeting efficiency, with a higher proportion of Luc-mRNA translation in the spleen compared to SA formulations. Figure 3 c).

[0108] 4. SM-102sLNPs exhibit higher lysosomal escape efficiency under the same cellular uptake conditions.

[0109] In DC2.4 cells, the two types of nanoparticles exhibited similar lysosomal colocalization behavior at the 1-hour time point (Cy5-mRNA colocalized with LysoTracker signaling). Figure 4 a). However, the colocalization of SM-102sLNPs with lysosomes was significantly reduced at 3 and 5 hours. Quantitative Pearson correlation analysis showed that SM-102sLNPs had a lower R value ( Figure 4 b) indicates that its lysosomal escape efficiency is higher. Furthermore, the fluorescence intensity distribution shows that SM-102s LNPs (red) and lysosomal signals (green) spatially separate after prolonged incubation. Figure 4 a).

[0110] Despite significant differences in lysosomal escape, flow cytometry and confocal microscopy analysis showed no statistically significant difference in cellular uptake between the two formulations at all time points (p>0.05). Figure 4 (c, d). This indicates that the lysosomal escape advantage of SM-102 is not due to differences in uptake capacity, but rather because its physicochemical properties are more conducive to endosome membrane disruption or intracellular transport pathway regulation.

[0111] Cytotoxicity assays (CCK-8 assay) showed no significant change in the survival rate of DC2.4 cells after treatment with mRNA concentrations ranging from 0 to 2000 ng / mL, and no difference between the two formulations. Figure 5 a). The apoptosis rate is also similar ( Figure 5 b).

[0112] 5. SM-102s LNPs 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 it does not excessively induce the expression of co-stimulatory molecules CD80, CD86 and MHC-II. Figure 6 bd, fh). This separation of antigen presentation from DC activation is accompanied by moderate activation of inflammatory signals: IL-1β and IL-6 mRNA levels are lower than in the MC3 group ( Figure 6 The activity of the TLR4-NF-κB pathway was lower in the TLR4 group than in the MC3 group (the levels of TLR4, MyD88, p-p65, and cytokine proteins were lower in the TLR4 group than in the MC3 group). Figure 6 (kq). This indicates that SM-102 can moderately promote DC maturation and inflammatory responses without overactivating MyD88-dependent TLR4-NF-κB signaling, while simultaneously enhancing antigen processing / stability. This functional difference makes SM-102sLNPs more advantageous in controlled immunization applications.

[0114] 6. SM-102sLNPs-OVA induces stronger systemic and antigen-specific T cell responses.

[0115] according to Figure 6 Following immunization of mice with regimen a (dose 0.5 mg / kg), CD4 levels in peripheral blood and spleen of mice in the SM-102sLNPs-OVA group were [data missing]. + and CD8 + The proportion of T cells was higher in the MC3 group than in the MC3 group. Figure 7 (b, c) OVA tetramer staining showed that antigen-specific CD8+ was present in the spleen and blood of the SM-102 group. + A higher proportion of T cells ( Figure 7 (d, e) suggests that it may enhance MHC-I antigen presentation or cytotoxic T cell cross-activation. H&E staining showed no significant pathological damage in any organs. Figure 8 This indicates good safety.

[0116] 7. SM-102sLNPs-OVA inhibits tumor growth and metastasis by enhancing T cell infiltration and inducing apoptosis.

[0117] In B16F10-OVA tumor-bearing mice ( Figure 9 In a), the tumor volume of the SM-102 group ( Figure 9 bd) and weight ( Figure 9 e) significantly lower than the PBS and MC3 groups, with no difference in body weight ( Figure 9 f). H&E and TUNEL staining of tumor tissue showed decreased cell density and increased apoptosis in the SM-102 group. Figure 9 g, h). Flow cytometry and immunohistochemistry confirmed that CD45 was present in the tumor microenvironment of the SM-102 group. + White blood cells, CD8a + and CD4 + T cell infiltration was significantly increased. Figure 10 ae), and the expression of CD69, a marker of T cell activation, is increased ( Figure 11 a, b).

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

[0119] In summary, this invention achieves "low inflammation-high immune activation" by adjusting lipid composition. Figure 14 The molecular mechanisms of this invention (such as the correlation between inhibition and activation of specific signaling pathways) are explored. These lipid nanoparticles exhibit a specific mode of action in splenic APCs that enhances antigen presentation and T cell activation, making them suitable for cancer immunotherapy (such as activating tumor-specific cytotoxic T cells and inhibiting immune escape from the tumor microenvironment). In summary, the key points of this invention include:

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

[0121] (2) Innovative design of spleen-targeted delivery system: improve the translation efficiency of mRNA in the spleen at low dose, enhance the presentation ability of antigen in spleen APCs, and activate tumor-specific toxic T cells.

[0122] (3) Balance between immune activation and inflammatory homeostasis: By optimizing the inflammatory properties of LNPs, the contradiction of "excessive inflammatory suppression efficacy" in traditional LNPs can be resolved.

[0123] (4) Synergistic enhancement of efficacy and safety: Achieving highly efficient anti-tumor immune responses (such as T cell activation and proliferation) at low doses, avoiding the toxic side effects of traditional LNPs. Applicable to cancer immunotherapy and providing a new paradigm for the development of other mRNA nanomedicines.

[0124] Although the technical solution of the present invention has 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 solution and technical effects of the present invention, and should not be regarded as any limitation on the scope of protection of the present invention. Simple modifications, alterations, or improvements made based on the technical concept of the present invention are all within the scope of protection claimed 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-PEG2000, and long-chain saturated fatty acids are (17.5-25): (3.5-5): (13.5-19): (0.5-0.75): (50-65). The long-chain saturated fatty acid is palmitic acid.

2. The lipid nanoparticles according to claim 1, characterized in that: 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 nanoparticles according to claim 1, characterized in that: 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.

4. The use of spleen-targeting lipid nanoparticles as described in any one of claims 1-3 in the preparation of mRNA vaccines.

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 immunostimulatory protein or a chemokine.

6. The application according to claim 4, characterized in that: The application includes, but is not limited to, one or more of the following aspects: (1) Application in the preparation of mRNA tumor vaccines that reduce the activation of the TLR4 / MyD88 / NF-κB pathway; (2) Application in the preparation of mRNA tumor vaccines that enhance spleen targeting and delivery efficiency; (3) Application in the preparation of mRNA tumor vaccines that enhance antigen presentation and immune response; (4) Application in the preparation of mRNA tumor vaccines that enhance anti-tumor efficacy; (5) Application in the preparation of mRNA tumor vaccines with low toxicity and dosage 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 MyD88-dependent TLR4-NF-κB signaling, moderately promoting DC maturation and inflammatory response, and enhancing antigen processing / stability; And / or, the improvement of spleen targeting and delivery efficiency includes, but is not limited to, improving mRNA translation efficiency in the spleen and improving mRNA expression in the spleen; And / or, the enhancement of antigen presentation and immune response includes, but is not limited to, enhancing lysosomal escape, promoting endosome membrane fusion, increasing mRNA cytoplasmic release efficiency, increasing DC cell antigen expression, and activating antigen-specific CD8. + T cells enhance MHC-I antigen presentation; And / or, the improvement in 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 dosage flexibility include, but are not limited to, improving dendritic cell survival, reducing organ pathological damage, and reducing clinical dosage.

8. A spleen-targeting mRNA tumor vaccine, characterized in that: The mRNA tumor vaccine comprises spleen-targeting lipid nanoparticles as described in any one of claims 1-3 and mRNA encoding tumor antigens encapsulated therein.

9. The mRNA tumor vaccine according to claim 8, characterized in that: The tumor antigens include, but are not limited to, tumor-associated antigens, tumor-specific antigens, and virus-associated antigens. And / or, the size of the mRNA tumor vaccine is 40-200 nm.

10. A method for preparing a spleen-targeting mRNA tumor vaccine as described in any one of claims 8-9, characterized in that: The preparation method includes the following steps: SM-102, cholesterol, DSPC, DMG-PEG 2000 and long-chain saturated fatty acids were dissolved in ethanol to obtain an organic phase; The mRNA encoding the tumor antigen was dissolved in citrate buffer to obtain the aqueous phase; The organic phase and the aqueous phase were mixed at a volume ratio of (2-4):1, and the resulting purified product was a spleen-targeted mRNA tumor vaccine.

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