Lipid nano-particles based on modified natural saponin, preparation method and application of lipid nano-particles

Abstract

The invention relates to the technical field of biological materials, in particular to lipid nanoparticles based on modified natural saponin, a preparation method and application of the lipid nanoparticles, and the modified natural saponin comprises natural saponin modified by metal complexes and / or natural saponin modified by targeting molecules. The natural saponin is selected from at least one of glycyrrhizic acid, aescin, soyasaponin, ginsenoside, ursolic acid and oleanolic acid. The modified natural saponin can effectively simulate the structural function of cholesterol compounds in lipid nanoparticles, can overcome the problems of unstable physicochemical properties of cholesterol, potential safety hazards of sources and the like, and can significantly improve the gene transfection and silencing efficiency of nucleic acid drugs as a drug carrier. And a new solution is provided for gene therapy and vaccine development. The preparation method of the lipid nanoparticles based on the modified natural saponin is mild in reaction condition, simple and convenient to operate, easy to control and beneficial to industrial production.

Description

Technical Field

[0001] This invention relates to the field of biomaterials technology, and in particular to lipid nanoparticles based on modified natural saponins, their preparation methods, and their applications. Background Technology

[0002] Gene therapy aims to deliver target nucleic acid molecules to specific target cells to treat diseases by correcting or compensating for gene defects. Gene therapy has shown great potential in both hereditary and acquired diseases. However, nucleic acid drugs, due to their anionic nature and sensitivity to endonucleases, often require gene vectors for effective delivery. Currently, gene vectors are mainly divided into two categories: viral vectors and non-viral vectors. While viral vectors have high in vivo transfection efficiency, their application is limited by issues such as immunogenicity, potential carcinogenicity, and capacity limitations. Non-viral vectors, on the other hand, have attracted widespread attention due to their high safety and ease of modification, but their delivery efficiency still needs improvement.

[0003] Four-component lipid nanoparticles (LNPs) are important non-viral delivery carriers in gene therapy, mainly consisting of ionizable lipids, auxiliary lipids (such as DSPC or DOPE), cholesterol, and PEG-lipids. Cholesterol enhances the stability of LNPs by filling the gaps between lipid molecules to maintain the integrity of the bilayer structure and regulating membrane fluidity to balance rigidity and flexibility. However, cholesterol may cause problems such as cytotoxicity, insufficient targeting ability, and limited delivery efficiency. For example, high-dose LNPs may cause liver damage due to cholesterol metabolic burden, and traditional formulations have poor targeting of extrahepatic tissues.

[0004] To optimize the performance of LNPs, research has shifted towards novel alternative materials. For example, natural saponins with steroidal structures, due to their anticancer activity and biocompatibility, can serve as cholesterol substitutes to reduce toxicity and enhance function, but problems such as low gene transfection rate and silencing efficiency still exist. Summary of the Invention

[0005] This invention aims to at least solve one of the technical problems existing in related technologies. Therefore, the first objective of this invention is to provide lipid nanoparticles based on modified natural saponins; the second objective is to provide a method for preparing lipid nanoparticles based on modified natural saponins; and the third objective is to provide applications of lipid nanoparticles based on modified natural saponins.

[0006] To achieve the first objective, the technical solution adopted by this invention is as follows: Lipid nanoparticles based on modified natural saponins, wherein the modified natural saponins include natural saponins modified with metal complexes and / or natural saponins modified with targeting molecules; the general structural formula of the modified natural saponins is shown below: or ; Where: SAPOs represent natural saponins; TMs represent targeted molecules; MCMs represent metal complex modules; SAPOs are selected from at least one of glycyrrhizic acid, aescin, daidzein, ginsenoside, ursolic acid and oleanolic acid. TMs are selected from at least one of folic acid, RGD peptide and cRGD peptide; Among them, RGD peptide is a short peptide formed by sequentially linking arginine, glycine and aspartic acid, and cRGD peptide is a short peptide formed by sequentially linking cyclic arginine, glycine and aspartic acid. The molecular structural formulas of MCMs are shown below: ; Wherein, M represents a coordinating metal ion, selected from any one of zinc, calcium, magnesium, iron, aluminum, copper, europium, and pyroxene; m represents the number of CH2 atoms in the alkyl chain, selected from an integer between 3 and 20; Y represents a coordinating atom, selected from any one of O, N, and S; Z 1 and Z 2 Z represents a pyridine cyclic group or a polynitrogen cyclic group. 1 and Z 2 Each is independently selected from any one of dimethylpyridinium, adenine, quinoline, and cyclotinophene. R represents amino or hydroxyl.

[0007] Furthermore, when the modified natural saponins include natural saponins modified using metal complexes and natural saponins modified using targeted molecules, the two types of modified natural saponins are mixed in a mass ratio of 1:10 to 10:1.

[0008] Furthermore, it also includes at least one of cationic lipids, phospholipids, and polyethylene glycol-modified lipids.

[0009] Furthermore, the cationic lipid is selected from at least one of (2,3-dioleoxypropyl)trimethylammonium chloride, N-[1-(2,3-dioleoxy)propyl]-N,N,N-trimethylammonium chloride, and (6Z,9Z,28Z,31Z)-heptadec-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butyrate.

[0010] Furthermore, the phospholipid is selected from at least one of 1,2-dioleoyl-sn-propanetriyl-3-phosphatidylethanolamine, 1,2-distearate-sn-glycerol-3-phosphocholine, and 1,2-dioleoyl-sn-glycerol-3-phosphocholine.

[0011] Furthermore, the PEGylated lipid is selected from 1,2-dimyristoyl-sn-glycerol-3-methoxy polyethylene glycol-2000 and / or 1,2-distearate-sn-glycerol-3-phosphate ethanolamine-polyethylene glycol-2000.

[0012] To achieve the second objective, the technical solution adopted by this invention is as follows: A method for preparing lipid nanoparticles based on modified natural saponins, used to prepare lipid nanoparticles based on modified natural saponins as described in any of the above-mentioned methods. Modification of natural saponins using metal ions includes the following steps: a1. Natural saponins, carboxyl activating reagents and N-hydroxysuccinimide are added to a polar organic solvent in a molar ratio of 1:1:1 to 1:10:10. The mixture is stirred at 35 to 70°C for 3 to 10 hours. Then, a compound containing a coordination unit and an organic base are added. The mixture is stirred at 25 to 90°C for 36 to 54 hours to obtain saponin-ligand conjugates. The coordination unit includes pyridine, carboxylic acid, or mercapto groups that can coordinate with metal ions; a2. Mix the saponin-ligand conjugate with a compound containing metal ions in equal molar amounts and stir for 0.5–12 h to obtain modified natural saponins containing metal complexes. Modification using targeted molecules, natural saponins, includes the following steps: b1. The target molecule, carboxyl activator and N-hydroxysuccinimide are added to a polar organic solvent in a molar ratio of 1:1:1 to 1:10:10. The mixture is stirred at 35 to 70 °C for 3 to 10 h. Then ethylenediamine and a basic catalyst are added. The mixture is stirred at 25 to 90 °C for 12 to 36 h to obtain the target molecule-ethylenediamine conjugate. b2. Natural saponins, carboxyl activating reagents and N-hydroxysuccinimide are added to a polar organic solvent in a molar ratio of 1:1:1 to 1:10:10. The mixture is stirred at 35 to 70°C for 3 to 10 hours. Then, the targeting molecule-ethylenediamine coupling agent and organic base are added. The mixture is stirred at 25 to 90°C for 36 to 54 hours to obtain modified natural saponins containing the targeting molecule.

[0013] Furthermore, the preparation process of lipid nanoparticles is as follows: Cationic lipids, auxiliary lipids, polyethylene glycol-modified lipids, and modified natural saponins were dissolved in an organic solvent, and the organic solvent was removed by rotary evaporation to obtain a thin film. After hydration, the film was incubated at 30–70°C for 30 min–2 h and then ultrasonically vibrated to obtain lipid nanoparticles.

[0014] To achieve the third objective, the technical solution adopted by this invention is as follows: Applications of lipid nanoparticles based on modified natural saponins, as described in any of the preceding claims, include their use as drug carriers.

[0015] Furthermore, the drug includes nucleic acid drugs.

[0016] The above-described one or more technical solutions in the embodiments of the present invention have at least one of the following technical effects: This invention provides lipid nanoparticles based on modified natural saponins, their preparation method, and their applications. Modified natural saponins can effectively mimic the structure and function of cholesterol in lipid nanoparticles, and overcome the problems of cholesterol's unstable physicochemical properties and potential safety hazards related to its source. Using them as drug carriers can significantly improve the gene transfection and silencing efficiency of nucleic acid drugs, thus providing a new solution for gene therapy and vaccine development. Furthermore, the preparation method provided by this invention features mild reaction conditions, simple operation, and easy control, which is beneficial for industrial production.

[0017] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0018] Figure 1 This is a graph showing the relationship between LNPs, particle size, and PDI of different modified natural saponins provided in Example 1 of the present invention.

[0019] Figure 2 This is a graph showing the relationship between LNPs and Zeta potential for different modified natural saponins provided in Example 1 of this invention.

[0020] Figure 3 This is the NMR spectrum of GA-BS provided in Embodiment 6 of the present invention.

[0021] Figure 4 This is the NMR spectrum of GA-RGD provided in Embodiment 6 of the present invention.

[0022] Figure 5 This is a diagram showing the experimental results of transfection of HeLa and other cells by different nanocomposites provided in Example 7 of the present invention.

[0023] Figure 6This is a diagram showing the experimental results of transfection of HepG2 and RAW264.7 cells with different nanocomposites provided in Example 7 of this invention.

[0024] Figure 7 This is a diagram showing the experimental results of gene silencing with different nanocomposites provided in Example 7 of the present invention. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention. The following embodiments are used to illustrate this invention, but cannot be used to limit the scope of this invention.

[0026] In the following embodiments, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used are commercially available, unless otherwise specified, and are carried out in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions.

[0027] Example 1 Lipid nanoparticles (LNPs) based on modified glycyrrhizic acid, consisting of folic acid-modified glycyrrhizic acid (GA-FA) and / or Zn. 2+ Glycyrrhizic acid (GA-Zn-DPA) modified with coordinated metal complexes serves as a cholesterol substitute, in conjunction with DOTAP, DOPE, and DMG-PEG. 2000 It is assembled from multiple parts.

[0028] I. The structural formula of GA-FA is shown below: The preparation process is as follows: At room temperature, folic acid (FA) (1 mmol, 0.45 g) was dissolved in DMSO (20 mL), and then EDC·HCl (1.2 mmol, 0.24 g) and NHS (2 mmol, 0.24 g) were added. The mixture was heated to 50 °C and stirred for 6 h. After cooling to room temperature, ethylenediamine (0.6 g, 10 mmol) and pyridine (0.25 mL) were added and the mixture was stirred for 24 h. The mixture was then precipitated with acetonitrile, filtered, and the residue was washed three times with diethyl ether and dried under vacuum to obtain a deep yellow folic acid-ethylenediamine conjugate (FA-NH2). Glycyrrhizic acid (GA) (3.5 mmol, 2.9 g) was dissolved in DMSO (15 mL) at room temperature. Then, EDC·HCl (4.2 mmol, 0.81 g) and NHS (7 mmol, 0.81 g) were added. The mixture was heated to 50 °C and stirred for 6 h. After cooling to room temperature, triethylamine (1 mL) and the previously prepared FA-NH2 (0.7 mmol, 0.33 g) were added sequentially. After stirring for 48 h, the mixture was precipitated with acetonitrile, filtered, and the solvent was removed by rotary evaporation. The filtrate was then dried under vacuum to obtain GA-FA.

[0029] DOTAP, DOPE, DMG-PEG 2000 GA-FA was dissolved in a mixed solvent of chloroform and methanol (volume ratio of chloroform to methanol 1:1) at a molar ratio of (40-50):(30-40):(20-30):(10-20). The organic solvent was removed by rotary evaporation, and the mixture was placed in a vacuum oven to allow complete evaporation of the organic solvent, yielding a lipid nanoparticle film. This film was then hydrated with pure water, incubated at 60°C for 1 hour, and followed by ultrasonic oscillation for 1 hour to obtain a series of GA-FA-LNPs, such as... Figure 1 A and Figure 2 As shown in A, its particle size and Zeta potential parameters meet the requirements of an ideal gene vector.

[0030] II. The structural formula of GA-Zn-DPA is shown below: The preparation process is as follows: Glycyrrhizic acid (2.05 mmol, 1.69 g) was dissolved in DMSO (10 mL), and EDC·HCl (0.495 mmol, 94.78 mg) and NHS (0.82 mmol, 94.84 mg) were added. The mixture was stirred at 50 °C for 6 h. After cooling to room temperature, triethylamine (400 μL) and the coordination unit N-[4-[[(5-hydroxypentyl)amino]methyl]benzyl]-N-(pyridin-2-methyl)amine DxDA-AP (0.41 mmol, 166.6 mg) were added sequentially. The mixture was stirred at room temperature for 48 h. The product was purified by rapid chromatography and dried to obtain GA-DPA. At room temperature, GA-DPA and zinc nitrate hexahydrate were mixed in equimolar amounts and stirred for 0.5 h to obtain the metal ion coordination compound GA-Zn-DPA. DOTAP, DOPE, DMG-PEG 2000GA-Zn-DPA was dissolved in a mixed solvent of chloroform and methanol (volume ratio of 1:1) at a molar ratio of (20-30):(30-40):(20-40):(5-10). The organic solvent was removed by rotary evaporation, and the solution was placed in a vacuum oven to allow complete evaporation of the organic solvent, yielding a lipid nanoparticle film. This film was then hydrated with pure water, incubated at 60°C for 1 hour, and followed by ultrasonic oscillation for 1 hour to obtain a series of GA-Zn-DPA-LNPs, such as... Figure 1 B and Figure 2 As shown in B, its particle size and Zeta potential parameters meet the requirements of an ideal gene vector.

[0031] DOTAP, DOPE, DMG-PEG 2000 GA-Zn-DPA and GA-FA were dissolved in a mixed solvent of chloroform and methanol (volume ratio of 1:1) at a molar ratio of (30-40):(10-20):(20-30):(20-30):(5-10). The organic solvent was removed by rotary evaporation, and the solution was placed in a vacuum oven to allow complete evaporation of the organic solvent, yielding a lipid nanoparticle film. This film was then hydrated with pure water, incubated at 60°C for 1 hour, and followed by ultrasonic oscillation for 1 hour to obtain a series of GA-FA+GA-Zn-DPA-LNPs, such as... Figure 1 C and Figure 2 As shown in C, its particle size and Zeta potential parameters meet the requirements of an ideal gene vector.

[0032] The particle size of the above-mentioned modified natural saponin LNPs can be controlled below 200 nm, the PDI is below 0.3, and the zeta potential is above 20 mV. These parameters all meet the requirements of an ideal drug delivery carrier.

[0033] Example 2 LNPs based on modified soybean saponin Ba, specifically soybean saponin Ba modified with RGD peptide (Ba-RGD) and / or Zn. 2+ Coordinated metal complexes modified with soybean saponin Ba (Ba-Zn-Qu) serve as cholesterol substitutes, in conjunction with DOTAP, DOPE, and DMG-PEG. 2000 It is assembled from multiple parts.

[0034] I. The structural formula of Ba-RGD is shown below: The preparation process is as follows: At room temperature, soybean saponins (958.5 mg), EDC·HCl (230 mg), and NHS (230 mg) were dissolved in DMSO (10 mL). The mixture was heated to 50 °C and stirred for 6 h. Then, the mixture was cooled to room temperature, and triethylamine (320 μL) and RGD peptide (69.3 mg) were added. After stirring for 48 h, the mixture was precipitated with acetonitrile, filtered, and the solvent was removed by rotary evaporation. The filtrate was then dried under vacuum to obtain Ba-RGD.

[0035] DOTAP, DOPE, DMG-PEG 2000 Ba-RGD was dissolved in a mixed solvent of chloroform and methanol in a molar ratio of 40:30:20:10 (the volume ratio of chloroform to methanol was 1:1). The organic solvent was removed by rotary evaporation, and the mixture was placed in a vacuum oven to allow the organic solvent to evaporate completely, resulting in a lipid nanoparticle film. The film was then hydrated with pure water, incubated at 60°C for 1 hour, and then sonicated for 1 hour to obtain Ba-RGD-LNPs.

[0036] II. The structural formula of Ba-Zn-Qu is shown below: The preparation process is as follows: At room temperature, soybean saponins (958.5 mg), EDC·HCl (230 mg), and NHS (230 mg) were dissolved in DMSO (10 mL). The mixture was heated to 50 °C and stirred for 6 h. Then, the mixture was cooled to room temperature, and triethylamine (320 μL) and 8-quinolinyl-4-[2-(5-hydroxypentanamino)acetamido]benzenesulfonamide (72.2 mg) containing quinoline (Qu) were added sequentially. After stirring at room temperature for 48 h, the product was purified by rapid chromatography and dried to obtain Ba-Qu. At room temperature, Ba-Qu was mixed with zinc nitrate hexahydrate in equimolar amounts and stirred for 2 hours to obtain the metal ion coordination compound Ba-Zn-Qu. DOTAP, DOPE, DMG-PEG 2000 Ba-Zn-Qu was dissolved in a mixed solvent of chloroform and methanol in a molar ratio of 30:40:20:10 (the volume ratio of chloroform to methanol was 1:1). The organic solvent was removed by rotary evaporation, and the mixture was placed in a vacuum oven to allow the organic solvent to evaporate completely, resulting in a lipid nanoparticle film. The film was then hydrated with pure water, incubated at 60°C for 1 hour, and then sonicated for 1 hour to obtain Ba-Zn-Qu-LNPs.

[0037] DOTAP, DOPE, DMG-PEG 2000Ba-Zn-Qu and Ba-RGD were dissolved in a mixed solvent of chloroform and methanol in a molar ratio of 30:20:20:20:10 (the volume ratio of chloroform to methanol was 1:1). The organic solvent was removed by rotary evaporation, and the mixture was placed in a vacuum oven to allow the organic solvent to evaporate completely, resulting in a lipid nanoparticle film. The film was then hydrated with pure water and incubated at 60°C for 1 hour, followed by ultrasonic oscillation for 1 hour to obtain Ba-Zn-Qu+Ba-RGD-LNPs.

[0038] Example 3 LNPs based on modified aescin, namely aescin modified with cRGD peptide (aescin-cRGD) and / or Zn. 2+ Aescin-Zn-A, modified with coordinated metal complexes, serves as a cholesterol substitute, in conjunction with DOTAP, DOPE, and DMG-PEG. 2000 It is assembled from multiple parts.

[0039] I. The structural formula of aescin-cRGD is shown below: The preparation process is as follows: At room temperature, aescin (1.13 g), EDC·HCl (230 mg) and NHS (230 mg) were dissolved in DMSO (10 mL). The mixture was heated to 50 °C and stirred for 6 h. After cooling to room temperature, triethylamine (320 μL) and cRGD peptide (120.7 mg) were added. After stirring for 48 h, the mixture was precipitated with acetonitrile, filtered, and the solvent was removed by rotary evaporation. The filtrate was then dried under vacuum to obtain aescin-cRGD.

[0040] DOTAP, DOPE, DMG-PEG 2000 Aescin-cRGD was dissolved in a mixed solvent of chloroform and methanol (volume ratio of chloroform to methanol was 1:1) at a molar ratio of 40:30:20:10. The organic solvent was removed by rotary evaporation, and the mixture was placed in a vacuum oven to allow the organic solvent to evaporate completely, resulting in a lipid nanoparticle film. The film was then hydrated with pure water, incubated at 60°C for 1 hour, and then sonicated for 1 hour to obtain aescin-cRGD-LNPs.

[0041] II. The structural formula of aescin-Zn-A is shown below: The preparation process is as follows: At room temperature, aescin (1.13 g), EDC·HCl (230 mg), and NHS (230 mg) were dissolved in DMSO (10 mL). The mixture was heated to 50 °C and stirred for 6 h. Then, the mixture was cooled to room temperature, and triethylamine (320 μL) and N-[2-(adenine-9-yl)ethyl]-N-(2-phenylethyl)-2-[(5-hydroxypentyl)amino]acetamide (72.2 mg) containing the adenine (A) coordination unit were added sequentially. After stirring at room temperature for 48 h, the product was purified by rapid chromatography and dried to obtain aescin-A. At room temperature, aescin-A and zinc nitrate hexahydrate were mixed in equimolar amounts and stirred for 2 hours to obtain the metal ion coordination compound aescin-Zn-A. DOTAP, DOPE, DMG-PEG 2000 Aescin-Zn-A was dissolved in a mixed solvent of chloroform and methanol (volume ratio of chloroform to methanol was 1:1) at a molar ratio of 20:30:40:10. The organic solvent was removed by rotary evaporation, and the mixture was placed in a vacuum oven to allow the organic solvent to evaporate completely, resulting in a lipid nanoparticle film. The film was then hydrated with pure water and incubated at 60°C for 1 hour, followed by ultrasonic oscillation for 1 hour to obtain aescin-Zn-A-LNPs.

[0042] DOTAP, DOPE, DMG-PEG 2000 Aescin-Zn-A and aescin-cRGD were dissolved in a mixed solvent of chloroform and methanol in a molar ratio of 40:10:20:20:10 (the volume ratio of chloroform to methanol was 1:1). The organic solvent was removed by rotary evaporation, and the mixture was placed in a vacuum oven to allow the organic solvent to evaporate completely, resulting in a lipid nanoparticle film. The film was then hydrated with pure water and incubated at 60°C for 1 hour, followed by ultrasonic oscillation for 1 hour to obtain aescin-Zn-A + aescin-cRGD-LNPs.

[0043] Example 4 LNPs based on modified glycyrrhizic acid, consisting of glycyrrhizic acid modified with cRGD peptide (GA-cRGD) and / or Zn. 2+ Glycyrrhizic acid (GA-Zn-Qu), modified with a coordinated metal complex (Zn-Qu), was used as a cholesterol substitute in combination with DOTAP, DOPE, and DMG-PEG, respectively. 2000 It is assembled from multiple parts.

[0044] The GA-Zn-Qu structure is as follows: The preparation process is as follows: At room temperature, 658.3 mg of glycyrrhizic acid, 153.4 mg of EDC·HCl, and 153.4 mg of NHS were mixed in 10 mL of DMSO and stirred at 50 °C for 6 h. Then, 72.2 mg of 8-quinolinyl-4-[2-(5-hydroxypentanamino)acetamido]benzenesulfonamide (Qu) containing the Qu coordination unit and 320 μL of triethylamine were added at room temperature, and the reaction was continued for 48 h. The product was purified by rapid chromatography to obtain GA-Qu with a yield of 51%.

[0045] At room temperature, GA-Qu was mixed with an equimolar amount of zinc nitrate hexahydrate and stirred for 2 hours to form the metal ion coordination compound soybean saponin GA-Zn-Qu.

[0046] DOTAP, DOPE, DMG-PEG 2000 GA-Zn-Qu was dissolved in a mixture of chloroform and methanol at a molar ratio of 40:30:20:10. The organic solvent was removed by rotary evaporation, and the mixture was placed in a vacuum oven to allow the organic solvent to evaporate completely. A lipid nanoparticle film was observed. The film was dissolved in pure water, incubated at 60°C for 1 hour, and then ultrasonically vibrated using an ultrasonic cleaner to obtain GA-Zn-Qu-LNPs.

[0047] The structural formula of GA-cRGD peptide is as follows: The preparation process is as follows: At room temperature, 822.9 mg glycyrrhizic acid, 191.7 mg EDC·HCl, and 191.7 mg NHS were dissolved in 10 mL DMSO and stirred at 50 °C for 6 h. Then, 320 μL of triethylamine and 69.3 mg of cRGD peptide were added at room temperature, and stirring was continued at 25 °C for 48 h. The mixture was then precipitated with acetonitrile, filtered, and the solvent was removed by rotary evaporation. The filtrate was then dried in a vacuum oven to obtain GA-cRGD peptide with a yield of 73%.

[0048] DOTAP, DOPE, DMG-PEG 2000 GA-cRGD peptides were dissolved in a mixture of chloroform and methanol at a molar ratio of 25:40:25:10. The organic solvent was removed by rotary evaporation, and the mixture was placed in a vacuum oven to allow the organic solvent to evaporate completely. A lipid nanoparticle film was observed. The film was dissolved in pure water, incubated at 60°C for 1 hour, and then ultrasonically vibrated using an ultrasonic cleaner to obtain GA-cRGD peptide-LNPs.

[0049] DOTAP, DOPE, DMG-PEG 2000GA-Zn-Qu and GA-cRGD peptides were dissolved in a mixture of chloroform and methanol at a molar ratio of 35:15:20:20:10. The organic solvent was removed by rotary evaporation, and the mixture was then placed in a vacuum oven to allow the organic solvent to evaporate completely. A lipid nanoparticle film was observed. The film was dissolved in pure water, incubated at 60°C for 1 hour, and then ultrasonically vibrated using an ultrasonic cleaner to obtain GA-Zn-Qu+GA-cRGD peptide-LNPs.

[0050] Example 5 LNPs based on modified aescin were derived from aescin and Zn modified with RGD peptide, respectively. 2+ Aescin, modified with a coordinated metal complex (aescinate-Zn-DPA), acts as a cholesterol substitute, in conjunction with DLin-MC3-DMA, DSPC, and DSPE-PEG. 2000 It is assembled from multiple parts.

[0051] The structural formula of aescin-RGD peptide is as follows: The preparation process is as follows: At room temperature, 1.13 g of aescin was dissolved in 10 mL of DMSO with 230 mg of EDC·HCl and 230 mg of NHS. The mixture was stirred at 50 °C for 6 h, and then 303.36 mg of N-[4-[[(5-hydroxypentyl)amino]methyl]benzyl]-N-(pyridin-2-methyl)amine (abbreviated as DxDA-AP) and 320 μL of triethylamine were added. The reaction was continued at 25 °C for 48 h. The product was purified by rapid chromatography to obtain aescin-DPA with a yield of 45%.

[0052] At room temperature, aescin-DPA was mixed with an equimolar amount of zinc nitrate hexahydrate and stirred for 2 hours to form Zn. 2+ The coordinated metal compound aescin-Zn-DPA.

[0053] DOTAP, DOPE, DMG-PEG 2000 Aescin-Zn-DPA was dissolved in a mixture of chloroform and methanol at a molar ratio of 40:30:20:10. The organic solvent was removed by rotary evaporation, and the mixture was placed in a vacuum oven to allow the organic solvent to evaporate completely. A lipid nanoparticle film was observed. The film was dissolved in pure water, incubated at 60°C for 1 hour, and then ultrasonically vibrated using an ultrasonic cleaner to obtain aescin-Zn-DPA-LNPs.

[0054] The structural formula of aescin-Zn-DPA is as follows: The preparation process is as follows: At room temperature, 1.13 g of aescin was dissolved in 10 mL of DMSO with 230 mg of EDC·HCl and 230 mg of NHS. The mixture was stirred at 50 °C for 6 h. Then, 320 μL of triethylamine and 346.34 mg of RGD peptide were added at room temperature, and the mixture was stirred for another 48 h. The mixture was then precipitated with acetonitrile, filtered, and the solvent was removed by rotary evaporation. The filtrate was then dried in a vacuum oven to obtain aescin-RGD peptide with a yield of 71%.

[0055] DOTAP, DOPE, DMG-PEG 2000 Aescin-cRGD peptide was dissolved in a mixture of chloroform and methanol at a molar ratio of 28:32:30:10. The organic solvent was removed by rotary evaporation, and the mixture was placed in a vacuum oven to allow the organic solvent to evaporate completely. A lipid nanoparticle film was observed. After complete dissolution with pure water, the film was incubated at 60°C for 1 hour and then ultrasonically vibrated using an ultrasonic cleaner to obtain aescin-RGD peptide-LNPs.

[0056] DOTAP, DOPE, DMG-PEG 2000 Aescin-Zn-A and aescin-cRGD peptide were dissolved in a mixture of chloroform and methanol at a molar ratio of 30:10:20:30:10. The organic solvent was removed by rotary evaporation, and the mixture was then placed in a vacuum oven to allow the organic solvent to evaporate completely. A lipid nanoparticle film was observed. The film was dissolved in pure water, incubated at 60 °C for 1 h, and then ultrasonically vibrated using an ultrasonic cleaner to obtain aescin-Zn-A + aescin-cRGD peptide-LNPs.

[0057] Example 6 Based on modified glycyrrhizic acid-LNPs, glycyrrhizic acid (GA-RGD) and Zn modified with RGD peptide were respectively... 2+ Glycyrrhizic acid (GA-Zn-BS) modified with a coordinated metal complex (Zn-BS) serves as a cholesterol substitute, in combination with DOTAP, DOPE, and DMG-PEG. 2000 It is assembled from multiple parts.

[0058] The GA-Zn-BS structure is as follows: The preparation process is as follows: Glycyrrhizic acid (2.05 mmol, 1.69 g) was dissolved in 10 mL DMSO at room temperature. EDC·HCl (0.495 mmol, 94.78 mg) and NHS (0.82 mmol, 94.84 mg) were added, and the mixture was stirred at 50 °C for 6 h. Then, 400 μL of triethylamine was added at room temperature, followed by the addition of the coordination unit N-[2-(adenine-9-yl)ethyl]-N-(2-phenylethyl)-2-[(5-hydroxypentyl)amino]acetamide (BS) (0.41 mmol, 471.52 mg) to the reaction mixture. The mixture was stirred at room temperature for another 48 h. The product was purified by rapid chromatography to obtain GA-BS, and its NMR spectrum is shown below. Figure 3 As shown, the yield is 35%.

[0059] At room temperature, GA-BS is mixed with an equimolar amount of zinc nitrate hexahydrate and stirred for 0.5-12 h to form the metal ion coordination compound GA-Zn-BS.

[0060] The structural formula of GA-RGD peptide is as follows: The preparation process is as follows: Glycyrrhizic acid (3.5 mmol, 2.9 g) was dissolved in 15 mL DMSO at room temperature. EDC·HCl (4.2 mmol, 0.81 g) and NHS (7 mmol, 0.81 g) were added, and the mixture was stirred at 50 °C for 6 h. Then, 1 mL of triethylamine was added at room temperature, along with RGD peptide (0.7 mmol, 242.44 mg). The mixture was stirred at room temperature for another 48 h. The product GA-RGD was then precipitated with acetonitrile, filtered, and the solvent was removed by rotary evaporation. After vacuum drying, the product GA-RGD was obtained, and its NMR spectrum is shown below. Figure 4 As shown, the yield is 70%.

[0061] DOTAP, DOPE, DMG-PEG 2000 GA-RGD was dissolved in a mixed solvent of chloroform and methanol in a molar ratio of 25:35:30:10 (the volume ratio of chloroform to methanol was 1:1). The organic solvent was removed by rotary evaporation, and the mixture was placed in a vacuum oven to allow the organic solvent to evaporate completely, resulting in a lipid nanoparticle film. The film was then hydrated with pure water, incubated at 60°C for 1 hour, and then ultrasonically vibrated for 1 hour to obtain GA-RGD-LNPs.

[0062] DOTAP, DOPE, DMG-PEG 2000GA-RGD and GA-BS were dissolved in a mixed solvent of chloroform and methanol in a molar ratio of 30:10:25:25:10 (the volume ratio of chloroform to methanol was 1:1). The organic solvent was removed by rotary evaporation, and the mixture was placed in a vacuum oven to allow the organic solvent to evaporate completely, resulting in a lipid nanoparticle film. The film was then hydrated with pure water and incubated at 60°C for 1 hour, followed by ultrasonic oscillation for 1 hour to obtain GA-RGD+GA-Zn-BS-LNPs.

[0063] Example 7 The preparation of the nucleic acid drug and the nanocomposite of lipid nanoparticles based on modified natural saponins is as follows: GA-RGD+GA-Zn-BS-LNPs and DNA prepared in Example 6 were diluted with sodium acetate / acetic acid buffer at pH 5.2 and then mixed in equal volumes. The mass ratio of GA-RGD+GA-Zn-BS-LNPs to DNA was 4:1. The mixture was allowed to stand at room temperature for 30 min to obtain the nucleic acid drug with GA-RGD+GA-Zn-BS-LNPs as the carrier. The DNA is selected from the green fluorescent protein particle pEGFP and is denoted as pDNA. The nanocomposite prepared above was used in cell transfection experiments, gene silencing experiments, and cytotoxicity experiments.

[0064] I. Cell transfection experiment, the procedure is as follows: The transfection efficiency of the nanocomplex on DNA was tested using the enhanced green fluorescent protein plasmid (pEGFP) as a reporter gene. HeLa cells were transfected at 2 × 10⁶ cells / day. 4 Cells were seeded at a density of 100 cells / well in 96-well plates and cultured for 24 hours until cell confluence reached 70%–80%. Transfection was then performed by adding GA-RGD+GA-Zn-BS-LNPs / DNA nanocomplex (0.25 μg DNA / well), mixing it with fresh culture medium, and replacing the original medium. Transfection continued for 48 hours. EGFP expression was observed and photographed using an inverted fluorescence microscope. The results are as follows: Figure 5 As shown, the transfection efficiency of the nanocomposite in cells was significantly higher than that of Lipofectamine 2000 (Lipo2000) as a pEGFP vector.

[0065] siRNA transfection experiments were performed using fluorescently labeled negative control siRNA (siRNA-NC-FAM) as a reporter gene. Logarithmic growth phase cells (HepG2 and RAW264.7 cells) were collected and transfected at a rate of 2 × 10⁻⁶ cells / year. 4Cells were seeded at a density of 100 cells / well in 96-well plates. After culturing for 24 hours until the cell confluence reached 70%–80%, the medium was replaced with fresh medium containing GA-FA+GA-Zn-BS-LNPs / siRNA nanocomplex (final siRNA concentration 0.14 μg / well). After transfection for another 24 hours, the medium was discarded, each well was washed twice with PBS, and fresh medium was added again. The fluorescence expression of transfected FAM-siRNA was observed and photographed using an inverted fluorescence microscope. The results are shown below. Figure 6 As shown, the transfection efficiency of modified nanoliposomes as carriers in both cell types was significantly higher than that of siRNA transfection efficiency when Lipo2000 was used as a carrier.

[0066] II. Gene silencing experiment, the procedure is as follows: The transfection efficiency of the nanocomplex on DNA was tested using an enhanced green fluorescent protein plasmid (pEGFP) as a reporter gene. A fluorescently labeled negative control siRNA (siRNA-NC-FAM) was used as a reporter gene for siRNA transfection experiments. Hela-EGFP and HepG2-EGFP cells were cultured at 2 × 10⁻⁶ cells / year. 4 Cells were seeded at a density of 100 cells / well in 96-well plates. When the cell density reached 70%–80%, fresh culture medium containing GA-RGD+GA-Zn-BS-LNPs / DNA-siRNA nanocomplex was added, with a siRNA-DNA concentration of 0.2 μM per well. After culturing for another 24 hours, gene silencing was observed and photographed using an inverted fluorescence microscope. The results are as follows: Figure 7 As shown in the figure, when GA-RGD+GA-Zn-BS-LNPs are used as vectors, they exhibit superior gene silencing efficiency in Hela-EGFP and HepG2-EGFP cells, significantly higher than the gene silencing efficiency of Lipo2000 as a vector and the unused material (blank control).

[0067] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Lipid nanoparticles based on modified natural saponins, characterized in that, The modified natural saponins include natural saponins modified with metal complexes and / or natural saponins modified with targeting molecules; the general structural formula of the modified natural saponins is shown below: or ; Where: SAPOs represent natural saponins; TMs represent targeted molecules; MCMs represent metal complex modules; SAPOs are selected from at least one of glycyrrhizic acid, aescin, daidzein, ginsenoside, ursolic acid and oleanolic acid. TMs are selected from at least one of folic acid, RGD peptide and cRGD peptide; Among them, RGD peptide is a short peptide formed by sequentially linking arginine, glycine and aspartic acid, and cRGD peptide is a short peptide formed by sequentially linking cyclic arginine, glycine and aspartic acid. The molecular structural formulas of MCMs are shown below: ； Wherein, M represents a coordinating metal ion, selected from any one of zinc, calcium, magnesium, iron, aluminum, copper, europium, and pyroxene; m represents the number of CH2 atoms in the alkyl chain, selected from an integer between 3 and 20; Y represents a coordinating atom, selected from any one of O, N, and S; Z 1 and Z 2 Z represents a pyridine cyclic group or a polynitrogen cyclic group. 1 and Z 2 Each is independently selected from any one of dimethylpyridinium, adenine, quinoline, and cyclotinophene. R represents amino or hydroxyl.

2. The lipid nanoparticles based on modified natural saponins as described in claim 1, characterized in that, When the modified natural saponins include natural saponins modified with metal complexes and natural saponins modified with targeted molecules, the two types of modified natural saponins are mixed in a mass ratio of 1:10 to 10:

1.

3. The lipid nanoparticles based on modified natural saponins as described in claim 1, characterized in that, It also includes at least one of cationic lipids, phospholipids, and polyethylene glycol-modified lipids.

4. The lipid nanoparticles based on modified natural saponins as described in claim 3, characterized in that, The cationic lipid is selected from at least one of (2,3-dioleoxypropyl)trimethylammonium chloride, N-[1-(2,3-dioleoxy)propyl]-N,N,N-trimethylammonium chloride, and (6Z,9Z,28Z,31Z)-heptadec-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butyrate.

5. The lipid nanoparticles based on modified natural saponins as described in claim 3, characterized in that, The phospholipid is selected from at least one of 1,2-dioleoyl-sn-propanetriyl-3-phosphatidylethanolamine, 1,2-distearate-sn-glycerol-3-phosphocholine and 1,2-dioleoyl-sn-glycerol-3-phosphocholine.

6. The lipid nanoparticles based on modified natural saponins as described in claim 3, characterized in that, The PEGylated lipids are selected from 1,2-dimyristoyl-sn-glycerol-3-methoxypolyethylene glycol-2000 and / or 1,2-distearate-sn-glycerol-3-phosphate ethanolamine-polyethylene glycol-2000.

7. A method for preparing lipid nanoparticles based on modified natural saponins, characterized in that, Used to prepare lipid nanoparticles based on modified natural saponins as described in any one of claims 1 to 6; Modification of natural saponins using metal complexes includes the following steps: a1. Natural saponins, carboxyl activating reagents and N-hydroxysuccinimide are added to a polar organic solvent in a molar ratio of 1:1:1 to 1:10:

10. The mixture is stirred at 35 to 70°C for 3 to 10 hours. Then, a compound containing a coordination unit and an organic base are added. The mixture is stirred at 25 to 90°C for 36 to 54 hours to obtain saponin-ligand conjugates. The coordination unit includes pyridine, carboxylic acid, or mercapto groups that can coordinate with metal ions; a2. Mix the saponin-ligand conjugate with a compound containing metal ions in equal molar amounts and stir for 0.5–12 h to obtain modified natural saponins containing metal complexes. Modifying natural saponins using targeted molecules includes the following steps: b1. The target molecule, carboxyl activator and N-hydroxysuccinimide are added to a polar organic solvent in a molar ratio of 1:1:1 to 1:10:

10. The mixture is stirred at 35 to 70 °C for 3 to 10 h. Then ethylenediamine and a basic catalyst are added. The mixture is stirred at 25 to 90 °C for 12 to 36 h to obtain the target molecule-ethylenediamine conjugate. b2. Natural saponins, carboxyl activating reagents and N-hydroxysuccinimide are added to a polar organic solvent in a molar ratio of 1:1:1 to 1:10:

10. The mixture is stirred at 35 to 70°C for 3 to 10 hours. Then, the targeting molecule-ethylenediamine coupling agent and organic base are added. The mixture is stirred at 25 to 90°C for 36 to 54 hours to obtain modified natural saponins containing the targeting molecule.

8. The method for preparing lipid nanoparticles based on modified natural saponins as described in claim 7, characterized in that, The preparation process of lipid nanoparticles is as follows: Cationic lipids, auxiliary lipids, polyethylene glycol-modified lipids, and modified natural saponins were dissolved in an organic solvent, and the organic solvent was removed by rotary evaporation to obtain a thin film. After hydration, the film was incubated at 30–70°C for 30 min–2 h and then ultrasonically vibrated to obtain lipid nanoparticles.

9. The application of lipid nanoparticles based on modified natural saponins, characterized in that, The lipid nanoparticles based on modified natural saponins as described in any one of claims 1 to 6, wherein the application includes use as a drug carrier.

10. The application of lipid nanoparticles based on modified natural saponins as described in claim 9, characterized in that, The drugs mentioned include nucleic acid drugs.

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