A method for preparing alkylated lentinan and its application in LNP vaccines.

Abstract

This invention belongs to the field of pharmaceutical formulation, drug delivery, and vaccine technology, and relates to a method for preparing alkylated lentinan and its application in lipid nanoparticle (LNP) vaccines. This invention modifies lentinan by alkylation, enabling it to dissolve efficiently in lipid nanoparticles (LNPs), making it suitable for the preparation of LNP mRNA vaccines. Using the alkylated lentinan of this invention to prepare mRNA vaccines allows for the co-loading / assembly of alkylated lentinan and mRNA into the same lipid nanoparticle delivery system, creating an integrated antigen and adjuvant vaccine system. This solves the problems in existing technologies, significantly enhances the strength and breadth of anti-tumor immune responses, and features high safety, efficient immune activation, and strong targeting, making it widely applicable for tumor prevention and treatment.

Description

Technical Field

[0001] This invention relates to the fields of pharmaceutical formulation, drug delivery and vaccine technology, and to a method for preparing alkylated lentinan and its application in LNP vaccines. Specifically, it relates to a drug-loaded lipid nanoparticle (LNP) constructed by synergistically combining messenger ribonucleic acid (mRNA) and alkylated lentinan as an immune adjuvant, and its application as a vaccine. Background Technology

[0002] Vaccines, by activating or enhancing the body's immune system's recognition and clearance of tumor-associated antigens / specific antigens, have become an important direction in tumor immunotherapy. mRNA vaccines, such as BioNTech's BNT111, can express tumor-associated antigens in vivo and are used to treat diseases such as advanced melanoma. However, using antigens alone often fails to induce a durable and potent immune response, and they usually need to be combined with adjuvants to enhance innate immune activation and antigen presentation efficiency.

[0003] However, existing liposome / lipoglycoside adjuvants still have room for improvement in terms of long-term biocompatibility, targeted distribution to tissues / immune organs, and adaptation to the tumor immune microenvironment. Meanwhile, bacterial polysaccharide vaccines typically face challenges such as limited immunogenicity and antigenic spectrum, and difficulty in compatibility with complex tumor antigen systems. Especially on the mRNA-LNP platform, a mature and reproducible solution is still lacking to achieve a synergistic integration of stable polysaccharide adjuvant exposure and efficient nucleic acid antigen encapsulation.

[0004] Polysaccharide vaccines already exist that utilize small fragments of bacterial capsular polysaccharides as antigens, such as the pneumococcal capsular polysaccharide CPS serotype-associated sugar antigen, which can exert a preventive effect against invasive pneumococcal disease (IPD). Pneumococcal capsular polysaccharide CPS can be used directly as a vaccine, acting as an immunogenic antigen to induce an immune response. There are also reports of using it directly as a vaccine after fatty acylation.

[0005] CN200910153754.X (publication date 2010-06-09) discloses the sulfation esterification of lentinan, achieving mild preparation and highly efficient antiviral activity of lentinan esters, suitable for industrial applications. CN201710416672.4 (publication date 2017-09-29) discloses the preparation of acetylated lentinan under specific conditions, solving the technical problem of improving the bioactivity of lentinan and achieving significantly improved antioxidant and antibacterial effects. However, these esterified lentinan derivatives cannot be used for the preparation of LNP.

[0006] Natural polysaccharide adjuvants, represented by lentinan (LNT), possess advantages such as a high safety margin, strong immune activation ability, and recognition by pattern recognition receptors, and have been used in the clinical treatment of tumors. Current research reports that monophosphate A combined with saponin QS-21 as a liposomal adjuvant, when co-formulated with recombinant protein antigens in shingles vaccines (such as Shingrix), can significantly enhance T-cell and antibody immune responses in the elderly, achieving better preventative effects.

[0007] However, how to efficiently integrate water-soluble macromolecular natural polysaccharides into the LNP vaccine system, utilize their pattern recognition function to exert a targeting effect, and prepare a vaccine system that integrates antigen and adjuvant is a thorny technical problem.

[0008] To address the aforementioned needs, this invention prepares alkylated lentinan with a substitution degree suitable for lipid nanoparticle (LNP) production via acylation. The alkylated lentinan is then co-loaded / assembled with mRNA into the same lipid nanoparticle delivery system, simultaneously achieving "adjuvant activation" and "antigen delivery" on the same particle, aiming to significantly enhance the strength and breadth of the anti-tumor immune response. This solves the problems of existing technologies lacking polysaccharide adjuvant systems with both high safety and efficient immune activation capabilities, leading to insufficient response strength and difficulty in obtaining stable and reproducible tumor prevention / treatment effects. It also addresses the limitations of traditional vaccines in terms of targeting, resulting in limited drug accumulation at the target site after systemic circulation and potential exposure to non-target organs (such as the liver) and safety risks. Summary of the Invention

[0009] This invention provides a method for preparing alkylated lentinan and its application in LNP vaccines. The alkylated lentinan is amphiphilic and can be used to form the lipid layer of LNP vaccines, with the lentinan encapsulated in a shell. This type of LNP exhibits both strong immunogenicity and good in vivo safety.

[0010] This invention provides a method for preparing alkylated lentinan, wherein lentinan is reacted with fatty acyl chlorides to obtain alkylated lentinan. The fatty acyl chlorides are selected from the group consisting of dodecyl chloride, tetradecyl chloride, hexadecyl chloride, or octadecyl chloride, preferably dodecyl chloride (lauroyl chloride) or hexadecyl chloride (palmitoyl chloride).

[0011] This invention provides a method for preparing alkylated lentinan, comprising adding a fatty acyl chloride to a lentinan solution, followed by purification and freeze-drying after the reaction is complete. The fatty acyl chloride is selected from the group consisting of dodecyl chloride, tetradecyl chloride, hexadecyl chloride, or octadecyl chloride, preferably dodecyl chloride (lauroyl chloride) or hexadecyl chloride (palmitoyl chloride). The solvent for the lentinan solution is selected from dimethyl sulfoxide and / or pyridine. Optionally, the lentinan solution further contains triethylamine and / or 4-dimethylaminopyridine (DMAP). The purification is performed by dialysis to remove unreacted reagents, small molecule byproducts, and further solvent.

[0012] This invention provides a method for preparing alkylated lentinan. The method includes: dissolving lentinan (LNT) in a mixed solvent of dimethyl sulfoxide and pyridine; adding triethylamine under stirring, and mixing thoroughly after the reaction; subsequently adding 4-dimethylaminopyridine (DMAP); then adding dodecyl chloride, and stirring at room temperature; after the reaction is complete, removing unreacted reagents and small molecule byproducts, and freeze-drying the resulting solution. The removal of unreacted reagents and small molecule byproducts is achieved by dialysis. The reaction solution is transferred to a dialysis bag and first dialyzed in dimethyl sulfoxide to remove unreacted reagents and small molecule byproducts. Then, it is transferred to pure water and dialyzed overnight for further purification; finally, the dialyzed solution is freeze-dried to obtain alkylated lentinan. The degree of substitution of the obtained alkylated lentinan is 0.03-0.28.

[0013] This invention provides a method for preparing alkylated lentinan vaccine LNPs: Ionizable lipids, auxiliary lipids, cholesterol, polyethylene glycol-modified lipids, and alkylated lentinan are dissolved in an organic solvent in a specific ratio to form the organic phase; a buffer containing mRNA is used as the aqueous phase. The organic and aqueous phases are mixed using a microfluidic or high-speed injection method to prepare LNPs loaded with mRNA. Ethanol is preferred as the organic solvent, and acetate buffer or phosphate buffer is preferred as the mRNA buffer. The microfluidic or high-speed injection method allows mixing of the organic and aqueous phases under high shear conditions, enabling lipid self-assembly to form LNP particles, and modifying the particle surface with lentinan. The modified particles can be recognized and bound by specific receptors on antigen-presenting cells such as dendritic cells, promoting the premature maturation and migration of antigen-presenting cells, and synergistically enhancing antigen processing, presentation, and T cell activation. The degree of substitution of alkylated lentinan is 0.1-0.25. When preparing the alkylated lentinan vaccine LNP, the mass ratio of mRNA to alkylated lentinan is 1:4-32, preferably 1:8-32, and more preferably 1:8-16.

[0014] This invention provides an alkylated lentinan vaccine LNP with the following features (one or more of which may be selected without conflict):

[0015] (1) Degree of hydroxyl substitution ≥ 0.1;

[0016] (2) The LNP particle size is approximately 80–180 nanometers;

[0017] (3) mRNA encapsulation rate ≥80%;

[0018] (4) The loading rate of lentinan on LNP is ≥30%.

[0019] This invention provides a method for preparing dodecylated lentinan, which is prepared through the following steps:

[0020] 1. Preparation: Dissolve lentinan in a mixed solvent of dimethyl sulfoxide and pyridine to obtain a lentinan solution; preferably, the volume ratio of dimethyl sulfoxide to pyridine is 1:1.

[0021] 2. Reaction: Triethylamine was added under stirring and stirred at room temperature for a period of time to ensure thorough mixing; then 4-dimethylaminopyridine was added and stirring continued; then fatty acyl chloride was added in proportion and stirred at 25-55℃ to achieve O-acylation hydrophobic modification; the preferred reaction temperature was 25℃ and the mass ratio of dodecyl chloride to LNT was 2 / 1.

[0022] 3. Purification: After the reaction is complete, the reaction solution is first dialyzed against dimethyl sulfoxide, and then transferred to pure water for dialyzed again. This is to remove unreacted reagents, small molecule byproducts, and solvents.

[0023] 4. Freeze-drying: Finally, the dialyzed solution was freeze-dried to obtain dodecyl lentinan (dLNT).

[0024] This invention uses nuclear magnetic resonance (NMR) or Fourier transform infrared spectroscopy (FT-IR) to confirm the introduction of alkyl chains (acyl characteristic peaks such as ester carbonyl groups around 1700 cm⁻¹); the degree of substitution (DS) is determined by elemental analysis or ¹H-NMR integration, DS = (integral value of the terminal methyl peak of dodecyl chloride / 3) / (integral value of the hydroxyl peak on the sugar ring); the polysaccharide content can be quantified by the phenol-sulfuric acid method to calculate the polysaccharide loading.

[0025] This invention provides an mRNA vaccine comprising a core structure and a surface structure. The core structure is an LNP core loaded with mRNA, and the surface structure is an alkylated lentinan anchored to the LNP phospholipid bilayer via hydrophobic fatty chains, with its hydrophilic polysaccharide chains exposed and covering the particle surface, thereby achieving dendritic cell receptor targeting and innate immune activation.

[0026] The LNP lipids of this invention contain ionizable lipids (such as SM-102), accessory lipids (such as DSPC), cholesterol, polyethylene glycol-modified lipids (such as DMG-PEG2000), and alkylated lentinan. SM-102 provides nucleic acid loading and endosome escape, DSPC enhances membrane stability, cholesterol regulates fluidity, polyethylene glycol-modified lipids provide steric stability and dispersibility, and alkylated lentinan enables dendritic cell receptor targeting and innate immune activation.

[0027] This invention provides a method for preparing alkylated lentinan vaccine LNP as follows:

[0028] Organic phase: Ionizable lipids, auxiliary lipids, cholesterol, polyethylene glycol-modified lipids, and alkylated lentinan are dissolved in ethanol;

[0029] Aqueous phase: dissolve mRNA in sodium acetate buffer at 25 mmol / L, pH 4;

[0030] Mixing: Microfluidics, impingement jet mixing, solvent injection, or thin-film hydration methods were used to induce self-assembly of the two phases at set flow rates and volume ratios. LNPs were prepared under the preferred conditions of an aqueous phase:organic phase volume ratio of 3:1 and a preferred total flow rate of 12 mL / min.

[0031] Purification: Ultrafiltration or dialysis removes free polysaccharides and residual solvents / impurities;

[0032] The alkylated lentinan vaccine LNP prepared by this invention was characterized by particle size, PDI, encapsulation efficiency and polysaccharide loading rate (the polysaccharide loading rate can be calculated by quantifying the free polysaccharides in the filtrate using the phenol-sulfuric acid method).

[0033] This invention provides an application of alkylated lentinan vaccine LNP. The alkylated lentinan of this invention can be used to prepare nucleic acid delivery products, and is particularly suitable for preparing glycosylated modified LNPs to activate antigen-presenting cells and induce cellular and humoral immune responses against tumor antigens.

[0034] The present invention discloses a method for preparing alkylated lentinan and its application in mRNA vaccines. Through an acylation reaction, lentinan is reacted with fatty acyl chlorides to obtain alkylated lentinan. Then, mRNA is encapsulated within the LNP core using microfluidic methods. Utilizing the amphiphilic structure formed by the alkylated lentinan, its hydrophobic fatty chains are anchored to the phospholipid bilayer of the LNP, while the hydrophilic polysaccharide chains are exposed on the outer surface of the particle, forming an integrated structure of "outer adjuvant – core antigen". Alkylated lentinan can bind to pattern recognition receptors (such as Dectin-1) on the surface of antigen-presenting cells (APCs), activating pathways such as NF-κB and Dectin-1, promoting APC maturation and migration. After uptake, the LNP releases mRNA intracellularly, expresses antigen proteins, and presents them through the MHC-I / II pathway, while simultaneously upregulating co-stimulatory molecules, thereby achieving a synergistic effect of "adjuvant activation – antigen delivery" in both time and space, significantly enhancing T cell responses.

[0035] This invention prepares alkylated lentinan with a substitution degree suitable for lipid nanoparticle (LNP) production via acylation. The alkylated lentinan is then co-loaded / assembled with mRNA into the same lipid nanoparticle delivery system, simultaneously achieving "adjuvant activation" and "antigen delivery" on the same particle, aiming to significantly enhance the strength and breadth of the anti-tumor immune response. This addresses the lack of polysaccharide adjuvant systems with both high safety and efficient immune activation capabilities in existing technologies, and the resulting insufficient response strength, making it difficult to obtain stable and reproducible tumor prevention / treatment effects. The alkylated lentinan LNP of this invention can enhance the targeting of traditional vaccines, increase drug accumulation at the target site after systemic circulation, and reduce the risk of exposure to non-target organs (such as the liver) and safety risks. Attached Figure Description

[0036] Figure 1 Preparation route of alkylated lentinan; The degree of substitution of dLNT is based on the degree of acylation of the R group OH;

[0037] Figure 2 Schematic diagram of antigen presentation activation after co-assembly of dLNT / LNP vaccines;

[0038] Figure 3 : 1H-NMR detection results of dLNT;

[0039] Figure 4 FT-IR detection results of dLNT;

[0040] Figure 5 Results of mRNA encapsulation efficiency detection for dLNT / LNP vaccines co-assembled with mRNA at different degrees of substitution;

[0041] Figure 6Results of polysaccharide loading in co-assembled dLNT / LNP vaccines prepared with different ratios of dLNT and mRNA;

[0042] Figure 7 TEM image (100 nm) of dLNT / LNP vaccine.

[0043] Figure 8 dLNT / LNP vaccines promote DC uptake; (Test results)

[0044] Figure 9 Results of in vitro BMDC maturation assay for dLNT / LNP vaccine promotion;

[0045] Figure 10 Results of in vitro mOVA transfection assay for dLNT / LNP vaccine promotion;

[0046] Figure 11 dLNT / LNP vaccine promotes targeted lymph node detection results in vivo. Detailed Implementation

[0047] The present application is further described below with reference to embodiments and examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the application. Furthermore, it should be understood that after reading the teachings of this application, those skilled in the art can make various alterations or modifications to the application, and these equivalent forms also fall within the protection scope of the appended claims.

[0048] This application provides a method for preparing alkylated lentinan and its application in mRNA vaccines, comprising alkylated lentinan and lipid nanoparticles, wherein the alkylated lentinan is inserted into the lipid layer of the lipid nanoparticles through lipophilic fatty chains; and the lipid nanoparticles are loaded with drugs.

[0049] In some embodiments, the fatty acyl chloride substitutes more than 0.1% of the glycosyl group; a high degree of substitution ensures the introduction of sufficient fatty chains onto the sugar chain, thereby endowing lentinan with appropriate lipophilicity, which is beneficial for its insertion into the lipid layer. At the same time, it is necessary to avoid over-modification leading to damage to the polysaccharide's bulk structure and loss of its ability to activate the Dectin-1 receptor. The degree of substitution can be directly calculated by comparing the ratio of the integral area of ​​the methyl signal in the introduced acyl group to the integral area of ​​the characteristic hydrogen on the polysaccharide ring using ¹H-NMR.

[0050] In some embodiments, the lentinan has a loading rate of over 30% on LNPs; the high loading rate ensures sufficient dendritic cell activation capacity, improves the efficiency of antigen presentation in the body, and avoids stability and safety issues that may result from over-modification.

[0051] In some embodiments, the natural polysaccharide adjuvant comprises one type of lentinan. Lipid nanoparticles co-assembled with alkylated lentinan can be recognized by specific receptors on the surface of antigen-presenting cells, such as Dectin-1 and TLR4, thereby promoting the activation capacity of antigen-presenting cells. Specifically, the lipid nanoparticles co-assembled with alkylated lentinan have a high affinity for the Dectin-1 receptor on the surface of antigen-presenting cells, significantly activating the maturation of antigen-presenting cells and promoting antigen presentation efficiency.

[0052] Furthermore, the natural polysaccharide adjuvant is lentinan, with a molecular weight of 300 kDa to 1000 kDa. The lipid nanoparticles formed by co-assembling with alkylated lentinan have the strongest binding ability to activate the Dectin-1 receptor on the surface of antigen-presenting cells, thus achieving the most efficient maturation of antigen-presenting cells.

[0053] In some embodiments, the drug comprises mRNA;

[0054] In some embodiments, the alkylated lentinan simultaneously possesses lipophilic fatty acid chains and hydrophilic sugar chains. When the alkylated lentinan is added to ethanol, during the mixing process of the aqueous and ethanol phases, the lipophilic fatty acid chains insert it into the lipid layer, thereby exposing the hydrophilic sugar chains to the LNP surface, ultimately resulting in the LNP shell being coated with lentinan.

[0055] This alkylated lentinan is stably coated on the surface of LNPs by inserting fatty chains into the lipid layer. It can effectively activate antigen-presenting cells by targeting and binding to specific receptors on the surface of antigen-presenting cells, thereby comprehensively enhancing the entire process of antigen processing, presentation, and T cell activation, and thus efficiently inducing the activation of downstream immune signaling pathways and promoting the body's immune response.

[0056] In some embodiments, the lipid nanoparticles contain lipids of ionizable types, including accessory lipids, polyethylene glycol-modified lipids, and cholesterol. A suitable ratio of these lipid compositions can form stable lipid nanoparticle structures, effectively encapsulating nucleic acid drugs and maintaining stability during in vivo circulation.

[0057] In some embodiments, the molar ratio of the ionizable lipid, auxiliary lipid, cholesterol, and PEGylated lipid is (48-52):(8-12):(36-40):(1.5-4.5).

[0058] In some embodiments, the ionizable lipid comprises SM-102.

[0059] In some embodiments, the auxiliary lipid comprises DSPC.

[0060] In some embodiments, the PEGylated lipid comprises DMG-PEG2000.

[0061] SM-102 has good nucleic acid loading capacity and endosome escape performance, DSPC can enhance the stability of LNP, cholesterol can regulate the fluidity of lipid membranes, and DMG-PEG2000 can provide steric stability.

[0062] In some embodiments, the molar ratio of SM-102, DSPC, cholesterol, and DMG-PEG2000 is (48-52):(8-12):(36-40):(1.5-4.5). This ratio ensures LNP stability.

[0063] In some embodiments, the lipid nanoparticles have a particle size of 100-200 nanometers, and the encapsulation efficiency of the nucleic acid drug is ≥80%. A suitable particle size range facilitates the circulation and targeted delivery of LNPs in vivo, while a high encapsulation efficiency ensures effective loading and delivery of the nucleic acid drug. Furthermore, the lentinan loading efficiency of this application is greater than 30%, as determined by the phenol-sulfuric acid method to detect the free polysaccharide concentration in the filtrate collected during ultrafiltration.

[0064] In some embodiments, the nucleic acid drug comprises mRNA.

[0065] Another aspect of this application provides the application of lipid nanoparticles co-assembled with the aforementioned dodecylated lentinan in the preparation of nucleic acid delivery products;

[0066] The nucleic acid delivery product includes an antigen-presenting cell activation product.

[0067] Another aspect of this application provides a method for preparing lipid nanoparticles based on dodecylated lentinan, comprising:

[0068] Lipid nanoparticles based on the co-assembly of dodecyl lentinan were prepared by mixing mRNA in an aqueous sodium acetate solution and then mixing it with ethanol containing dissolved lipids and dodecyl lentinan using microfluidic methods.

[0069] In some embodiments, the preparation of lipid nanoparticles based on alkylated lentinan co-assembly comprises: dissolving the ionizable lipid, the auxiliary lipid, the cholesterol, the polyethylene glycol-modified lipid, and the alkylated lentinan in ethanol as an organic phase;

[0070] Lipid nanoparticles formed by co-assembling dodecyl lentinan were prepared by mixing a sodium acetate solution of nucleic acid drugs as the aqueous phase with the organic phase and the aqueous phase.

[0071] In some embodiments, mixing the organic phase and the aqueous phase includes using one of microfluidic methods, impingement jet mixing, solvent injection, or thin-film hydration.

[0072] In some embodiments, the flow rate of the microfluidic method is 10-14 mL / min. For example, the flow rate is 10 mL / min, 11 mL / min, 12 mL / min, 13 mL / min, or 14 mL / min, and any value in between.

[0073] In some embodiments, after mixing, free polysaccharides and impurities are removed by ultrafiltration or dialysis.

[0074] This application also provides a method for preparing unmodified LNPs. An organic phase is prepared by dissolving ionizable lipids, cholesterol, auxiliary lipids, and polyethylene glycol-modified lipids in ethanol at a specific molar ratio. Simultaneously, a sodium acetate solution of a nucleic acid drug is used as the aqueous phase. The two phases are mixed under high-speed shear conditions using a microfluidic or high-speed injection method, causing the lipids to self-assemble into an LNP carrying the nucleic acid drug.

[0075] Another aspect of this application provides a method for co-assembling lipid nanoparticles with dLNTs; wherein the alkylated lentinan is inserted into the lipid layer via a fatty acid chain; the lipid nanoparticles have a core and a surface ligand-modified shell, the shell being composed of lentinan, and the core being composed of a nucleic acid drug and lipid components.

[0076] In this embodiment, lipid nanoparticles co-assembled with dLNTs undergo surface modification via the following process: Nucleic acid drugs are mixed in a sodium acetate solution. Utilizing the dual properties of dLNTs (lipophilic fatty chains and hydrophilic glycan chains), the fatty chains are inserted into the lipid layer while the glycan chains are exposed on the LNP surface. This successfully and stably modifies the polysaccharide onto the LNP surface, thereby constructing lipid nanoparticles with antigen-presenting cell activation function. The modified LNPs can specifically recognize and bind to antigen-presenting cell surface receptors (such as Dectin-1), achieving highly efficient activation of antigen-presenting cells. This activation mechanism significantly enhances the antigen-presenting capacity of dendritic cell antigen-presenting cells, promotes effective antigen presentation to T cells, and thereby activates the overall immune pathway, significantly improving the immunogenicity of the vaccine.

[0077] In tumor vaccine applications, lipid nanoparticles enter cells via endocytosis, followed by the efficient release of the encapsulated nucleic acid drugs through the endosome-lysosome pathway. The released nucleic acid drugs bind to targets in the cytoplasm or nucleus through specific molecular mechanisms, regulating gene expression or function, thereby achieving therapeutic effects such as gene silencing, tumor antigen protein expression, or gene editing. As a delivery carrier for nucleic acid drugs, LNPs exhibit precise gene regulation capabilities, making them particularly suitable for tumor vaccine development. By efficiently delivering nucleic acid sequences of tumor-associated antigens or immunostimulatory molecules, they significantly enhance the antigen presentation and immune activation capabilities of antigen-presenting cells, thereby stimulating a potent immune response against tumors and providing strong support for the immunotherapy of malignant tumors such as melanoma.

[0078] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this application, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0079] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0080] It should be understood that in the various embodiments of this application, the order of the above-mentioned processes does not imply the order of execution. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0081] Example 1 Preparation of alkylated lentinan

[0082] In this article, the abbreviations include: LNP: lipid nanoparticles; mRNA: messenger RNA; LNT: lentinan (purchased from Acmec, 308414651); dLNT: alkylated lentinan; SM-102: heptadecano-9-yl-8-((2-hydroxyethyl)(6-oxo-6-((undecyloxy)hexyl)amino)octanoate; DSPC: distearate phosphatidylcholine; DMG-PEG2000: dimyristoylglycerol-polyethylene glycol 2000.

[0083] 1. Effects of different temperatures and ratios of dodecyl chloride and lentinan on the degree of substitution of dLNT

[0084] 20 mg of lentinan (LNT) was dissolved in 2 mL of a mixed solvent of DMSO and pyridine (volume ratio 1:1). 36.5 mg of triethylamine was added under stirring, and the mixture was stirred for 5 minutes to ensure thorough mixing. Then, 7.5 mg of 4-dimethylaminopyridine (DMAP) was added, and the mixture was stirred for 5 minutes. Next, different masses of dodecyl chloride (1x: 29.5 mg, 2x: 59 mg, 4x: 88.5 mg, 8x: 177 mg) were added dropwise or added, and the mixture was stirred and reacted for 3 hours at different reaction temperatures of 25°C, 35°C, 45°C, and 55°C. After the reaction was complete, the reaction solution was transferred to a dialysis bag with a molecular weight cutoff of 8000-14000 Daltons. The bag was first dialyzed in 200-300 mL of DMSO for 6 hours to remove unreacted reagents and small molecule byproducts. Then, the bag was transferred to pure water and dialyzed overnight. Finally, the dialyzed solution was freeze-dried to obtain dLNT.

[0085] The introduction of alkyl chains was confirmed by ¹H-NMR or Fourier transform infrared spectroscopy (FT-IR) (acyl characteristic peaks, such as the ester carbonyl group around 1700 cm⁻¹). The peak area was calculated by ¹H-NMR, and the degree of substitution was calculated using the formula: DS = (integral value of the terminal methyl peak of dodecyl chloride / 3) / (integral value of the hydroxyl peak on the sugar ring). The product structure was characterized by FT-IR (confirming the ester carbonyl peak). The results of evaluating the differences in the degree of alkyl chain introduction and the variation of the degree of substitution under different temperatures and ratios are shown in Table 1. The degree of substitution of dLNT obtained in this application is 0.0322-0.2795. The degree of substitution of dLNT under the conditions of mass ratios of dodecyl chloride to LNT of 0.5 / 1, 1 / 1, and 2 / 1 at 25℃ is 0.0544±0.0027, 0.1122±0.0130, and 0.2439±0.0270, respectively, denoted as 0.05, 0.1, and 0.25 degree of substitution dLNT. Taking into account both reaction temperature and ratio, a mass ratio of 2:1 between dodecyl chloride and LNT at 25℃ was selected as the optimal reaction condition. Figure 3 , 4 The results of 1H-NMR and FT-IR detection of dLNT are presented.

[0086] Table 1: Degree of substitution of dLNT at different reaction temperatures and ratios

[0087] (All values ​​are the mean ± standard deviation for n=3)

[0088]

[0089] Example 2: Preparation of dLNT co-assembled lipid nanoparticles

[0090] SM-102 (0.53 mg), DSPC (0.12 mg), cholesterol (0.21 mg), and DMG-PEG2000 (0.17 mg) were dissolved in ethanol (125 μL) at a molar ratio of 50:10:35.5:4.5. Then, dLNT (0.16 mg) with different degrees of substitution (0.05, 0.1, 0.25) was added to form the ethanol phase. mRNA (19.8 μg) was dissolved in sodium acetate buffer (375 μL) at 25 mmol / L as the aqueous phase. LNPs were prepared by mixing the aqueous phase and organic phase at a volume ratio of 3:1 at a total flow rate of 12 mL / min using a herringbone microfluidic chip. After the reaction, the mixture was dialyzed in PBS (1×, pH 7.4) for 2 hours using a Slide-A-LyzerMINI dialysis unit with a MWCO of 3500 Daltons to remove ethanol and neutralize the system. After the reaction, the system was filtered through a 0.22 μm sterile filter membrane to remove uncoupled ligands and impurities, yielding dLNT / LNP. The particle size and potential of dLNT / LNP with different dLNT to mRNA ratios were determined using a particle size analyzer. The encapsulation efficiency of dLNT / LNP with different dLNT to mRNA ratios was determined using a Ribogreen kit. Simultaneously, the polysaccharide loading rate of dLNT / LNP with different dLNT to mRNA ratios was determined using the phenol-sulfuric acid method. The results are listed in Table 2. Figure 5 The encapsulation efficiency of dLNTs with a substitution degree of 0.1 was 71.33%, and that of dLNTs with a substitution degree of 0.25 was 85.00%. Figure 6 The loading rate of alkylated lentinan (dLNT) was significantly better than that of unalkylated lentinan (LNT). The mass ratio of mRNA to dLNT was 1:2-32. A mass ratio of 1:4 achieved a loading rate of 66.02%, a mass ratio of 1:8 achieved a loading rate of 88.41%, and a mass ratio of 1:16 achieved a loading rate of 85.21%. When preparing dLNT co-assembled lipid nanoparticles, the mass ratio of mRNA to dLNT was 1:4-32, preferably 1:8-32, more preferably 1:8-16. A ratio of 1:8 was selected as the optimal ratio, and further testing and efficacy verification were conducted.

[0091] Table 2: Mean particle size, mRNA encapsulation efficiency, and polysaccharide loading of dLNT / LNP formulations with different mRNA to dLNT ratios (mean ± standard deviation for n=3).

[0092]

[0093] Example 3: TEM detection of dLNT co-assembled lipid nanoparticles

[0094] The LNPs formed by the co-assembly of the prepared dLNTs were negatively stained and observed using transmission electron microscopy (TEM). The results are as follows: Figure 7 As shown in the image (scale bar is 100 nm). TEM images reveal that the LNPs exhibit a spherical nanostructure with clear edges and uniform morphology. The particle size is concentrated within approximately 100 nm, and there is no obvious aggregation, indicating that the prepared LNPs have good dispersibility and structural stability. These experimental results confirm that dLNT / LNPs possess the typical spherical morphology and good dispersion stability of lipid nanoparticles, maintaining structural integrity in TEM, demonstrating their suitability for in vivo nucleic acid drug delivery carrier applications.

[0095] Example 4: Flow cytometry detection of dLNT co-assembled lipid nanoparticles DC2.4 cell uptake.

[0096] To verify the effect of dLNT-prepared co-assembled lipid nanoparticles on promoting mRNA uptake by DC2.4 cells, flow cytometry was used for detection. First, in the dLNT / LNP preparation, Cy5 fluorescently tagged mRNA was loaded into the particles using microfluidic technology. Then, the Cy5-mRNA-loaded dLNT / LNP (or control LNP, dose 1 μg mRNA / well) was incubated with DC2.4 cells at four different time points: 0, 2, 4, and 6 hours. After incubation, the supernatant was discarded, and the cells were washed 2-3 times with PBS, digested with 0.25% trypsin-EDTA for 2 minutes, neutralized, centrifuged, and resuspended in PBS. The mean fluorescence intensity of Cy5 was measured by flow cytometry, with the untreated group serving as a negative control. Results are as follows: Figure 8 As shown, the Cy5 fluorescence intensity in the dLNT / LNP group increased significantly over time and was significantly higher than that in the LNP group, confirming that this modification strategy effectively enhances intracellular mRNA uptake and has potential in vaccine delivery.

[0097] Example 5: Experiment on stimulation of mouse bone marrow-derived dendritic cells by dLNT co-assembled lipid nanoparticles.

[0098] To evaluate the stimulatory effect of co-assembled lipid nanoparticles based on dLNT on the maturation of mouse bone marrow-derived dendritic cells, the co-assembled lipid nanoparticles prepared by dLNT were co-incubated with mouse bone marrow-derived dendritic cells for 24 hours. Then, the cells were administered at high and low doses of lentinan at concentrations of 100 and 50 μg / mL, respectively. The expression levels of maturation markers (CD80 and CD86) on the surface of mouse bone marrow-derived dendritic cells were detected by flow cytometry.

[0099] The results are as follows Figure 9As shown in the figure, experimental data revealed that dLNT co-assembled lipid nanoparticles significantly upregulated the expression of CD80 and CD86 on the surface of mouse bone marrow-derived dendritic cells with increasing concentration gradients. Specifically, dLNT / LNP increased the expression levels of CD80 and CD86 by up to 10%. These results indicate that dLNT co-assembled lipid nanoparticles can effectively stimulate the maturation of mouse bone marrow-derived dendritic cells, significantly enhance their antigen-presenting capacity, and provide strong support for the induction of subsequent immune responses.

[0100] Example 6: dLNT co-assembled lipid nanoparticles promote the translation of mOVA antigen protein

[0101] To evaluate the effectiveness of dLNT / LNP in antigen delivery, Western blotting was used to detect the expression of OVA model antigen proteins mediated by dLNT / LNP in dendritic cells. BMDC cells were transfected with LNP and dLNT / LNP containing 1 μg / mL mOVA, and after 24 hours of culture, cells were collected, total protein was extracted, and Western blotting was performed to detect OVA protein.

[0102] The results are as follows Figure 10 As shown in the figure. Western blot analysis revealed that dLNT / LNP significantly increased OVA protein expression in dendritic cells compared to the unmodified LNP group. At the tested dose, the OVA expression level in the dLNT / LNP group was approximately 6 times that of the unmodified group, demonstrating that dLNT co-assembled lipid nanoparticles can enhance mRNA transfection efficiency and antigen expression in dendritic cells.

[0103] Example 7: dLNT co-assembled LNP nanoparticles promote in vivo lymph node targeted detection

[0104] To evaluate the ability of co-assembled LNPs prepared by dLNT to promote lymph node targeting in mice, an intramuscular injection experiment was conducted in a 6–8 week old C57BL / 6 mouse model. LNPs and dLNT / LNPs were administered intramuscularly at high and low doses, respectively, with a loading of 0.5 and 1 mg / kg mLuc. Six hours after administration, 150 μg of potassium fluorescein (15 mg / mL, 100 mL volume) was injected intraperitoneally into the mice. In vivo bioluminescence imaging was performed 10 minutes later, followed by ex vivo imaging of major organs and lymph nodes.

[0105] The results are as follows Figure 11 As shown, dLNT co-assembled LNP nanoparticles can effectively enhance lymph node targeting ability and significantly improve mRNA transfection at lymph nodes.

[0106] This application belongs to the field of nanomedicine delivery technology, and relates to a method for synthesizing alkylated lentinan and its application in mRNA vaccines. The alkylated lentinan, by introducing alkyl fatty chains, acquires lipophilicity and can spontaneously insert into the phospholipid bilayer of lipid nanoparticles, thereby forming a polysaccharide shell on the LNP surface, significantly enhancing its activation ability for antigen-presenting cells. The introduced lentinan can specifically target and bind to antigen-presenting cell surface receptors (such as Dectin-1), achieving highly efficient activation of antigen-presenting cells, thereby promoting effective antigen presentation to T cells, significantly improving antigen presentation efficiency and enhancing the in vivo immune response, ultimately exerting immune efficacy and achieving both preventive and therapeutic effects.

Claims

1. A method for preparing alkylated lentinan for LNP vaccine, wherein lentinan is reacted with fatty acyl chloride to obtain alkylated lentinan, characterized in that, The reaction temperature is 25-55℃, the mass ratio of fatty acyl chloride to lentinan is 2:1, the fatty acyl chloride is selected from dodecyl chloride, tetradecyl chloride, hexadecyl chloride or octadecyl chloride, and the degree of substitution of the alkylated lentinan is 0.1-0.

25.

2. The production method according to claim 1, characterized by, Fatty acyl chloride was added to a lentinan solution, and after the reaction was completed, the solution was purified and freeze-dried to obtain the alkylated lentinan. The purification process involved dialyzing the alkylated lentinan obtained after the reaction with dimethyl sulfoxide and then transferring it to pure water for further dialysis.

3. The preparation method according to claim 1, characterized in that, The reaction solvent is selected from dimethyl sulfoxide and / or pyridine; optionally, the lentinan solution also contains triethylamine and / or 4-dimethylaminopyridine (DMAP).

4. Alkylated lentinan for LNP vaccines prepared by any one of the methods described in claims 1-3.

5. A preparation method of an alkylated lentinan LNP vaccine, comprising the alkylated lentinan according to claim 4, characterized in that, Ionizable lipids, auxiliary lipids, cholesterol, polyethylene glycol-modified lipids, and the alkylated lentinan were dissolved in an organic solvent in a certain proportion to form an organic phase; using a buffer containing mRNA as the aqueous phase, LNPs loaded with mRNA were prepared by microfluidic or high-speed injection methods; the organic solvent was ethanol, and the buffer for mRNA was acetate buffer or phosphate buffer.

6. The production method according to claim 5, wherein The molar ratio of mRNA to alkylated lentinan is 1:(8-16).

7. The preparation method according to claim 5, characterized in that, The molar ratio of the ionizable lipids, auxiliary lipids, cholesterol, and PEGylated lipids is (48-52):(8-12):(36-40):(1.5-4.5).

8. The preparation method according to claim 5, characterized in that, The ionizable lipid is SM-102, the auxiliary lipid is DSPC, and the PEGylated lipid is DMG-PEG2000.

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