A zwitterionic polypeptide lipid molecule and its application

Abstract

The present invention discloses a zwitterionic polypeptide lipid molecule and its application, belonging to the technical field of drug delivery system. The general formula of the zwitterionic polypeptide lipid molecule is R-(E-K) n , wherein R is a fatty acid, a diglyceride derivative, or a cholesterol derivative, E is glutamic acid, K is lysine, and n is an integer between 5 and 20. The zwitterionic polypeptide lipid molecule is formed by bonding a fatty acid, a diglyceride derivative, or a cholesterol derivative to an EK polypeptide. It can be used as an alternative component to PEG lipids in lipid nanoparticles, and can be combined with cationic lipids, phospholipids, and cholesterol to form lipid nanoparticles with the ability to deliver mRNA to cells and animals. Experimental verification shows that compared with PEG lipids, the lipid nanoparticles composed of the zwitterionic polypeptide lipid molecules provided by the present invention have improved cell transfection ability in vivo and in vitro, improved the delivery efficiency of the lipid nanoparticles, and have no obvious cytotoxicity.

Description

Technical Field

[0001] The present invention relates to the technical field of drug delivery systems, and in particular to a zwitterionic polypeptide lipid molecule and applications thereof. Background Art

[0002] Lipid nanoparticles (LNPs) are a delivery system for hydrophobic or hydrophilic molecular drugs, used for drug therapy and vaccine preparation. Drugs such as small molecules, nucleic acids, and proteins need to be delivered to specific locations to be effective. For example, oncology drugs need to be delivered to tumor tissue, and nucleic acid-based vaccines need to be delivered to cells for expression. Lipid nanoparticles have been proven to be effective in delivering small molecule drugs, nucleic acids, and proteins. For example, LNP vaccines that deliver mRNA have been widely studied, and some products have received clinical approval.

[0003] Lipid nanoparticles are generally composed of cationic lipids, phospholipids, cholesterol, and polyethylene glycol lipid molecules. Among them, polyethylene glycol (PEG) is used to improve the in vivo stability of nanoparticles and prolong their circulation time in the body, but PEG inevitably brings some problems. Studies have shown that PEG has certain immunogenicity, is easily cleared by the immune system, and causes allergic reactions (Polymers 2020, 12, 298). Ju et al. reported that the LNP vaccines currently used in clinical practice for SARS-CoV-2, such as BNT162b2 and mRNA-1273, will cause systemic immune responses in the human body, and this phenomenon is related to the PEG lipid component (ACS Nano, 2022, 16, 8, 11769–11780). Therefore, the development of a new lipid molecule with low immunogenicity, reduced immune side effects, and replacing the PEG lipid component in LNP is an urgent problem that researchers in this field need to solve.

[0004] Currently, studies have reported on several materials that can replace PEG, such as polyoxazoline, poly(N-vinylpyrrolidone), polyglycerol, and polyacrylamide (Polymers, 2020, 12, 298; Advanced Drug Delivery Reviews, 2022, 180, 114079). These materials have anti-protein adsorption properties and good biocompatibility. However, these materials have certain limitations, such as the difficulty and high cost of polyoxazoline synthesis (Nanoscale, 2015, 7, 13671-13679) and the low biodegradability of polyglycerol and polyacrylamide (Polymers, 2020, 12, 298).

[0005] Zwitterionic materials can also be used as alternatives to PEG. Their equal positive and negative charges give them excellent resistance to protein adsorption, resulting in lower immunogenicity and longer in vivo circulation (Nano Today, 2014, 9, 10-16). Common zwitterionic materials include polycarboxybetaine (PCB), polysulfobetaine (PSB), and zwitterionic peptides. Compared to polymers, peptides have a well-defined structure and better biosafety. However, peptide-modified lipid nanoparticles often utilize a reaction between a thiol group and maleimide. The introduced maleimide has potential side effects, hindering in vivo application and clinical translation. Therefore, how to overcome these issues and develop a material that can effectively replace the PEG lipid component in lipid nanoparticles is a problem that those skilled in the art need to solve. Summary of the Invention

[0006] The purpose of the present invention is to provide a zwitterionic lipid molecule to replace the polyethylene glycol (PEG) component in lipid nanoparticles (LNPs) to prepare a drug delivery system for delivering nucleic acid molecules.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] The present invention provides a zwitterionic polypeptide lipid molecule, wherein the general formula of the zwitterionic polypeptide lipid molecule is R-(EK) n , wherein R is a fatty acid, a diglyceride derivative or a cholesterol derivative, E is glutamic acid, K is lysine, and n is an integer of 5-20.

[0009] The present invention modifies fatty acids, diglyceride fatty acid ester derivatives or cholesterol derivatives to increase the amount of zwitterionic polypeptide EK n The prepared zwitterionic peptide lipid molecules can self-assemble with other lipid molecules to form lipid nanoparticles, among which the zwitterionic peptide EK n It is stably modified on the surface of nanoparticles, giving the nanoparticles anti-protein adsorption properties.

[0010] The zwitterionic polypeptide EK n , consisting of alternating arrangements of negatively charged glutamic acid (E) and positively charged lysine (K). Preferably, n=8.

[0011] Furthermore, the fatty acid, diglyceride fatty acid ester derivative or cholesterol derivative is connected to the N-terminus of the polypeptide molecule by a chemical coupling method.

[0012] Preferably, the fatty acid is a fatty acid having 1 to 21 carbon atoms.

[0013] Preferably, the fatty acid is a saturated fatty acid or an unsaturated fatty acid having a carbon number of 10 to 21. More preferably, the fatty acid is stearic acid or oleic acid.

[0014] The diglycerol fatty acid ester derivative is a compound formed by derivatizing diglycerol fatty acid ester to form a group that can be chemically coupled to the N-terminus of a polypeptide molecule, and may be, but is not limited to, diglycerol succinate monoester.

[0015] The diglycerol fatty acid ester is produced by esterifying two fatty acids with glycerol. The fatty acid used to produce the diglycerol fatty acid ester can be a fatty acid with 1-21 carbon atoms. Preferably, the fatty acid is a saturated fatty acid or an unsaturated fatty acid with 10-21 carbon atoms. More preferably, the fatty acid is oleic acid.

[0016] The cholesterol derivative is a compound formed by derivatizing cholesterol to form a group that can be chemically coupled to the N-terminus of a polypeptide molecule, and may be, but is not limited to, cholesterol succinate monoester.

[0017] Specifically, the carboxyl end of fatty acid, diglyceride succinate monoester or cholesterol succinate monoester binds to the polypeptide molecule EK n The N-terminus is bonded via an amide bond.

[0018] Specifically, the structural formula of the zwitterionic polypeptide lipid molecule is any one of formulas (I) to (IV),

[0019]

[0020] The present invention provides a method for preparing the zwitterionic polypeptide lipid molecule by solid phase synthesis, the method comprising: firstly synthesizing the polypeptide (EK) on a solid phase resin by solid phase synthesis n , n is 5-20, and then fatty acids, diglyceride succinate monoester or cholesterol succinate monoester are added to cap the polypeptide, and then lysis solution is added to remove the solid phase resin and side chain protecting groups to obtain the zwitterionic polypeptide lipid molecule.

[0021] The present invention also provides the use of the zwitterionic polypeptide lipid molecules in preparing lipid nanoparticle carriers.

[0022] The invention provides a lipid nanoparticle carrier, which comprises cationic lipid molecules, phospholipids, cholesterol and zwitterionic polypeptide lipid molecules.

[0023] The cationic lipid molecules can be, but are not limited to: 1-octylnonyl 8-[(2-hydroxyethyl)[6-O-6-(undecanyloxy)hexyl]amino]-octanoate (trade name SM102), ((4-hydroxybutyl)azadialkyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (trade name ALC-0315), (2,3-dioleyloxypropyl)trimethylammonium chloride (trade name DOTAP), 4-(N,N-dimethylamino)butyric acid (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl ester (trade name Dlin-MC3-DMA).

[0024] The phospholipids may be, but are not limited to, distearoylphosphatidylcholine (DSPC), 1,2-dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), distearoylphosphatidylethanolamine (DSPE), dioleoylphosphatidylethanolamine (DOPE), and dimyristoylphosphatidylethanolamine (DMPE).

[0025] Preferably, the cationic lipid molecule is SM102.

[0026] Preferably, the phospholipid is DSPC.

[0027] Preferably, the molar ratio of the cationic lipid molecules, phospholipids, cholesterol and zwitterionic lipid molecules is 50:10:38.5:1.5.

[0028] The present invention provides the use of the lipid nanoparticle carrier in the preparation of nucleic acid drugs.

[0029] The application includes: adding cationic lipid molecules, phospholipids, cholesterol and zwitterionic polypeptide lipid molecules into an acidic buffer containing nucleic acid, self-assembling to form lipid nanoparticles encapsulating nucleic acid, and preparing the nucleic acid drug.

[0030] Specifically, cationic lipids, phospholipids, cholesterol, and zwitterionic peptide lipid molecules were dissolved in ethanol, and nucleic acids were dissolved in 40 mM sodium acetate buffer. The ethanol solution and sodium acetate buffer were mixed at a volume ratio of 1:3, vortexed thoroughly, dialyzed to remove the ethanol molecules, and filtered to remove aggregated and precipitated lipid carriers to obtain a zwitterionic peptide-modified lipid nanoparticle solution.

[0031] The nucleic acid drug prepared by the present invention can be used to deliver exogenous nucleic acid molecules into cells so that the exogenous nucleic acid molecules can be translated and expressed in the cells; it can also be used to deliver exogenous nucleic acid molecules into experimental animals so that the exogenous nucleic acid molecules can be translated and expressed in the animals.

[0032] The beneficial effects of the present invention are:

[0033] (1) The present invention provides a zwitterionic polypeptide lipid molecule, which is formed by bonding a fatty acid, a diglyceride derivative, or a cholesterol derivative to an EK polypeptide. n After forming lipid nanoparticles with cationic lipids, phospholipids and cholesterol, they have good particle size and particle size distribution. Lipid nanoparticles have the ability to deliver mRNA to cells and animals. Therefore, zwitterionic polypeptide lipid molecules R-EK n It can be used as an alternative component to PEG lipids in lipid nanoparticles to overcome the problems of PEG.

[0034] (2) Experimental results show that, compared with PEG lipids, the lipid nanoparticles composed of zwitterionic polypeptide lipid molecules provided by the present invention have significantly improved cell transfection ability in vivo and in vitro, improved the delivery efficiency of lipid nanoparticles, and have no obvious cytotoxicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Mass spectra of the EK peptide lipid molecules prepared in Examples 1-4, (a) Ste-EK8; (b) Ole-EK8; (c) Chol-EK8; (d) DOG-EK8.

[0036] Figure 2 The cytotoxicity of EK peptide lipid molecules at different concentrations.

[0037] Figure 3 Figure 2 is the particle size diagram of GFP-mRNA loaded lipid nanoparticles.

[0038] Figure 4 Cell transfection results of GFP-mRNA-loaded lipid nanoparticles, (a) fluorescence microscopy photos of cells after transfection; (b) cell transfection efficiency and cell activity after transfection.

[0039] Figure 5 Figure 2 is the particle size diagram of Luci-mRNA loaded lipid nanoparticles.

[0040] Figure 6 To investigate the particle size stability of Luci-mRNA loaded lipid nanoparticles in serum.

[0041] Figure 7 The results of in vivo transfection of Luci-mRNA-loaded lipid nanoparticles in mice, (a) In vivo fluorescence imaging photos after cell transfection; (b) Fluorescence quantification results. DETAILED DESCRIPTION

[0042] The present invention will be further described below in conjunction with specific examples. The following examples are only used to illustrate the present invention and are not intended to limit the scope of application of the present invention. Without departing from the spirit and essence of the present invention, modifications or replacements made to the inventive method, steps or conditions all fall within the scope of the present invention.

[0043] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available reagents and materials unless otherwise specified.

[0044] Compounds involved in the examples:

[0045] Fmoc-lys(boc)-Wang Resin (loading = 0.348 mmol / g), Fmoc-Glu(OtBu)-OH (CAS: 71989-18-9), and Fmoc-Lys(Boc)-OH (CAS: 71989-26-9) were purchased from Jier Biochemical.

[0046] Stearic acid (CAS: 57-11-4), oleic acid (CAS: 112-80-1), diolein (CAS: 25637-84-7), cholesterol succinate (CAS: 1510-21-0), and cholesterol (CAS: 57-88-5) were purchased from Anaiji Chemical.

[0047] SM102 was purchased from Sinobond, and DSPC and DMG-PEG-2k were purchased from Jiankai Technology.

[0048] The English abbreviations of the compounds involved in the examples are as follows:

[0049] DMF: N,N-dimethylformamide, HOBT: 1-hydroxybenzotriazole, HBTU: O-benzotriazole-tetramethyluronium hexafluorophosphate.

[0050] Example 1: Preparation of stearic acid-modified EK peptide lipid molecules

[0051] Stearic acid-modified EK peptide was synthesized according to the solid phase synthesis method in the literature (Angew.Chem.Int.Ed., 2020, 59, 22378-22381). Specifically, 200 mg of Fmoc-lys(boc)-Wang Resin was first added to the solid phase synthesis tube and anhydrous DMF was added to swell for 1 hour. After draining the DMF, a deprotecting agent (piperidine:DMF=1:4) was added to remove the Fmoc group at the N-terminus, and the success of the deprotection was detected by the ninhydrin test method. Fmoc-Glu(OtBu)-OH (0.696 mmol, 296 mg), HBTU (0.696 mmol, 264 mg) and HOBT (0.696 mmol, 94 mg) were then dissolved in a coupling agent (DMF:N-methylmorpholine=95:5) and added to the solid phase synthesis tube for 2 hours. Repeat the above coupling and deprotection steps, add Fmoc-Lys(Boc)-OH and Fmoc-Glu(OtBu)-OH in sequence, connect 7 lysines and 8 glutamic acids in sequence, and finally add stearic acid (0.696 mmol, 198 mg) to cap the end. After the coupling is completed, add the cleavage solution (water: triisopropylsilane: trifluoroacetic acid)

[0052] =2.5:2.5:95) to remove the side chain protecting groups, filter the organic phase, add icy ether to precipitate the polypeptide lipid molecules, and remove the ether by centrifugation to obtain a stearic acid-modified EK peptide (Ste-EK8) solid powder.

[0053] The mass spectrometry characterization of Ste-EK8 is shown in the figure below. Figure 1 As shown in (a), according to the mass spectrometry detection results, the m / z of the polypeptide lipid molecule Ste-EK8 is 2343.414, which is consistent with expectations.

[0054] Example 2: Preparation of oleic acid-modified EK peptide lipid molecules

[0055] The oleic acid-modified EK peptide was synthesized according to the solid phase synthesis method in the literature (Angew.Chem.Int.Ed., 2020, 59, 22378-22381). Specifically, 200 mg of Fmoc-lys(boc)-Wang Resin was first added to the solid phase synthesis tube and anhydrous DMF was added to swell for 2 hours. After draining the DMF, a deprotecting agent (piperidine:DMF=1:4) was added to remove the Fmoc group at the N-terminus, and the success of the deprotection was detected by the ninhydrin test method. Fmoc-Glu(OtBu)-OH (0.696 mmol, 296 mg), HBTU (0.696 mmol, 264 mg) and HOBT (0.696 mmol, 94 mg) were dissolved in a coupling agent (DMF:N-methylmorpholine=95:5) and added to the solid phase synthesis tube for 1 hour. The coupling and deprotection steps were repeated, followed by the addition of Fmoc-Lys(Boc)-OH and Fmoc-Glu(OtBu)-OH, connecting seven lysine residues and eight glutamic acids in sequence. Oleic acid (0.696 mmol, 195 mg) was then added for capping. Finally, the resin was shrunk with methanol, and a lysis buffer (water: triisopropylsilane: trifluoroacetic acid = 2.5:2.5:95) was added to remove the protecting groups. The organic phase was filtered, and glacial ether was added to precipitate the polypeptide lipid molecules. The ether was removed by centrifugation to obtain a solid powder of the oleic acid-modified EK peptide (Ole-EK8).

[0056] The mass spectrometry characterization of Ole-EK8 is shown in the figure below. Figure 1 As shown in (b), according to the mass spectrometry detection results, the m / z of the polypeptide lipid molecule Ole-EK8 is 2340.678, which is consistent with expectations.

[0057] Example 3: Preparation of EK peptide lipid molecules modified with cholesterol derivatives

[0058] Cholesterol derivative-modified EK peptide was synthesized according to the solid phase synthesis method in the literature (Angew.Chem.Int.Ed., 2020, 59, 22378-22381). Specifically, 200 mg of Fmoc-lys(boc)-WangResin was first added to the solid phase synthesis tube and anhydrous DMF was added to swell for 2 hours. After draining the DMF, a deprotecting agent (piperidine:DMF=1:4) was added to remove the Fmoc group at the N-terminus, and the success of the deprotection was detected by the ninhydrin test method. Fmoc-Glu(OtBu)-OH (0.696 mmol, 296 mg), HBTU (0.696 mmol, 264 mg) and HOBT (0.696 mmol, 94 mg) were dissolved in a coupling agent (DMF:N-methylmorpholine=95:5) and added to the solid phase synthesis tube for 1 hour. Repeat the above coupling and deprotection steps, add Fmoc-Lys(Boc)-OH and

[0059] Seven lysine residues and eight glutamic acids were sequentially linked using Fmoc-Glu(OtBu)-OH, and end-capped with cholesterol succinate (0.696 mmol, 339 mg). Finally, the resin was shrunk with methanol, and a lysis buffer (water: triisopropylsilane: trifluoroacetic acid = 2.5:2.5:95) was added to remove the protecting groups. The organic phase was filtered, and glacial ether was added to precipitate the polypeptide lipid molecules. The ether was removed by centrifugation to obtain a solid powder of the cholesterol derivative-modified EK peptide (Chol-EK8).

[0060] The mass spectrometry characterization of Chol-EK8 is shown in the figure below. Figure 1 As shown in (c), according to the mass spectrometry detection results, the m / z of the polypeptide lipid molecule Chol-EK8 is 2545.458, which is consistent with expectations.

[0061] Example 4: Preparation of EK peptide lipid molecules modified with glycerol dioleate derivatives

[0062] 1. First, synthesize glycerol dioleate succinate monoester. The specific synthesis method is as follows:

[0063] 5 g of glycerol dioleate (8.05 mmol), 1.61 g of succinic anhydride (16.1 mmol) and 2.46 g of 4-dimethylaminopyridine (16.1 mmol) were dissolved in 50 mL of dichloromethane. After overnight reaction, the mixture was extracted with 1 M dilute hydrochloric acid and saturated sodium chloride, respectively. The organic phase was collected and the organic solvent was removed by rotary evaporation to obtain a crude product of glycerol dioleate succinate monoester, which was then purified by silica gel chromatography (eluent: n-hexane: ethyl acetate = 20:1) to obtain a pure product of glycerol dioleate succinate monoester.

[0064] 2. Synthesize EK peptide modified with glycerol dioleate derivative according to the solid phase synthesis method in the literature (Angew.Chem.Int.Ed., 2020, 59, 22378-22381). Specifically, first add 200mg of Fmoc-lys(boc)-Wang Resin to the solid phase synthesis tube, add anhydrous DMF to swell for 2h. After draining the DMF, add a deprotecting agent (piperidine:DMF=1:4) to remove the Fmoc group at the N-terminus, and use the ninhydrin test method to detect whether the deprotection is successful. Fmoc-Glu(OtBu)-OH (0.696mmol, 296mg), HBTU (0.696mmol, 264mg) and HOBT (0.696mmol, 94mg) are dissolved in a coupling agent (DMF:N-methylmorpholine=95:5) and added to the solid phase synthesis tube for 1h. The coupling and deprotection steps were repeated, followed by the addition of Fmoc-Lys(Boc)-OH and Fmoc-Glu(OtBu)-OH to sequentially link seven lysine residues and eight glutamic acids. The residues were capped with glycerol dioleate succinate (0.696 mmol, 502 mg). Finally, the resin was shrunk with methanol, and a lysis buffer (water: triisopropylsilane: trifluoroacetic acid = 2.5:2.5:95) was added to remove the protecting groups. The organic phase was filtered, and glacial ether was added to precipitate the polypeptide lipid molecules. The ether was removed by centrifugation to obtain a solid powder of glycerol dioleate derivative-modified EK peptide (DOG-EK8).

[0065] 3. Mass spectrometry characterization of DOG-EK8 Figure 1 As shown in (d), according to the mass spectrometry results, the m / z of the polypeptide lipid molecule DOG-EK8 is 2800.953, which is in line with expectations. Test Example 1: Cytotoxicity of EK peptide lipid molecules

[0066] 1 × 10 4 BHK cells were plated for 24 hours and then treated with 2μM, 10μM, 50μM, or 250μM of DMG-PEG2k, Ste-EK8, Ole-EK8, or Chol-EK8, respectively. After 24 hours of incubation, the culture medium was removed and fresh culture medium containing 10% CCK8 was added. After incubation at 37°C for 1.5 hours, the absorbance of each well was measured at 450 nm using a microplate reader. Cell viability was calculated by calculating the ratio of the absorbance value of the drug-treated well to the blank control well.

[0067] like Figure 2As shown, with increasing material concentration, Ste-EK8 and Ole-EK8 exhibit no significant cytotoxicity. However, at concentrations above 50 μM, Chol-EK8 exhibits significant cytotoxicity. At concentrations above 250 μM, DMG-PEG2k exhibits significant cytotoxicity. Therefore, Ste-EK8 and Ole-EK8 possess a better safety profile than DMG-PEG2k and are more suitable for high-dose injections.

[0068] Test Example 2: Preparation of GFP-mRNA-loaded lipid nanoparticles

[0069] 1. Preparation method of lipid nanoparticles

[0070] SM-102, DSPC, cholesterol, and peptide lipid molecules (Ste-EK8, Ole-EK8, or Chol-EK8) were dissolved in ethanol at a molar ratio of 50:10:38.5:1.5. mRNA encoding green fluorescent protein (GFP) was dissolved in 40 mM sodium acetate buffer. The ethanol solution and sodium acetate buffer were mixed at a volume ratio of 1:3, vortexed thoroughly, and dialyzed using a 1 kDa molecular weight cutoff. The ethanol was then filtered through a 0.45 μm pore membrane to remove aggregated and precipitated lipid carriers to yield mRNA-loaded lipid nanoparticle solutions (Ste-EK-LNP / mGFP, Ole-EK-LNP / mGFP, or Chol-EK-LNP / mGFP).

[0071] In a control group, SM-102, DSPC, cholesterol, and DMG-PEG2k were dissolved in ethanol at a molar ratio of 50:10:38.5:1.5. GFP-encoding mRNA was dissolved in 40 mM sodium acetate buffer. Lipid nanoparticles were prepared using the above method to obtain lipid nanoparticles containing the PEG lipid component (PEG-LNP / mGFP).

[0072] 2. Particle Size Characterization of GFP-mRNA-Loaded Lipid Nanoparticles

[0073] Dilute the LNP sample (Chol-EK-LNP / mGFP, Ste-EK-LNP / mGFP, or Ole-EK-LNP / mGFP) to 0.16 mg / mL with phosphate buffer, take 1 mL of the sample and place it in the dedicated sample cell of the particle size analyzer. Perform dynamic light scattering test with the particle size analyzer to measure the hydration kinetic particle size and particle size distribution of the LNP, such as Figure 3 The experiment was repeated three times and the average value was taken. The particle size and particle size distribution data are shown in Table 1.

[0074] Table 1 Particle size and size distribution of GFP-mRNA loaded lipid nanoparticles

[0075] lipid nanoparticles Particle size (nm) Particle size distribution PDI Chol-EK-LNP / mGFP 249.4±13.8 0.16 Ste-EK-LNP / mGFP 159.9±3.9 0.14 Ole-EK-LNP / mGFP 143.2±1.2 0.17

[0076] The results showed that the LNP particle size was between 100-300 nm and could be used for drug delivery in vivo and in vitro.

[0077] 3. Verify the effect of lipid nanoparticle delivery of mRNA at the cellular level

[0078] 2.5 × 10 4 After 24 hours, PEG-LNP / mGFP, Ste-EK-LNP / mGFP, Ole-EK-LNP / mGFP, or Chol-EK-LNP / mGFP containing 2 μg of GFP mRNA was added to 300 μL of serum-free DMEM medium. The culture medium in the 48-well plate was replaced. GFP expression was observed 24 hours later, and transfection efficiency was determined by flow cytometry.

[0079] The cell transfection results and transfection efficiency of LNP / mGFP are as follows Figure 4 As shown in the Figure 3, compared with traditional PEG components, LNPs containing zwitterionic peptide lipids have improved transfection efficiency and no obvious cytotoxicity.

[0080] Test Example 3: Preparation of Luci-mRNA-loaded lipid nanoparticles

[0081] 1. Preparation method of lipid nanoparticles

[0082] SM-102, DSPC, cholesterol, and peptide lipid molecules (Ste-EK8, Ole-EK8, or Chol-EK8) were dissolved in ethanol at a molar ratio of 50:10:38.5:1.5. Luciferase (Luci) mRNA was dissolved in 40 mM sodium acetate buffer. The ethanol solution and sodium acetate buffer were mixed at a volume ratio of 1:3, vortexed thoroughly, and dialyzed to remove the ethanol using a 1 kDa molecular weight cutoff. The aggregated and precipitated lipid carriers were then filtered through a 0.45 μm pore membrane to obtain mRNA-loaded lipid nanoparticle solutions (Ste-EK-LNP / mLuci, Ole-EK-LNP / mLuci, or Chol-EK-LNP / mLuci).

[0083] Simultaneously, a control group was set up in which SM-102, DSPC, cholesterol, and DMG-PEG were dissolved in ethanol at a molar ratio of 50:10:38.5:1.5. The mRNA encoding Luci was dissolved in 40 mM sodium acetate buffer. Lipid nanoparticles were prepared using the above method to obtain lipid nanoparticles containing the PEG lipid component (PEG-LNP / mLuci).

[0084] 2. Particle Size Characterization of Luci-mRNA-Loaded Lipid Nanoparticles

[0085] Dilute the LNP sample (Chol-EK-LNP / mLuci, Ste-EK-LNP / mLuci, or Ole-EK-LNP / mLuci) to 0.16 mg / mL with phosphate buffer, take 1 mL of the sample and place it in the dedicated sample cell of the particle size analyzer. Perform dynamic light scattering test with the particle size analyzer to measure the hydration kinetic particle size and particle size distribution of the LNP, such as Figure 5 The experiment was repeated three times and the average value was taken. The particle size and particle size distribution data are shown in Table 2.

[0086] Table 2 Particle size and size distribution of Luci-mRNA loaded lipid nanoparticles

[0087]

[0088]

[0089] The results showed that the particle size of LNPs was less than 300 nm and could be used for drug delivery in vivo or in vitro.

[0090] 3. Particle size stability of Luci-mRNA-loaded lipid nanoparticles

[0091] The Luci-mRNA-loaded LNP samples (PEG-LNP / mLuci, Chol-EK-LNP / mLuci, Ste-EK-LNP / mLuci, or Ole-EK-LNP / mLuci) were diluted with complete culture medium containing 10% fetal bovine serum and placed in a 37°C shaking incubator to simulate the in vivo environment. Dynamic light scattering tests were performed using a particle size analyzer at 0 h, 2 h, 4 h, 8 h, 18 h, 24 h, and 48 h to monitor the hydration kinetics and particle size changes of the LNPs, as shown in Figure 3. Figure 6 shown.

[0092] The results showed that LNP could maintain particle size stability within 48 hours of incubation in serum-containing culture medium, indicating that it still had good stability after replacing PEG lipids with EK peptide lipid molecules.

[0093] 4. Verify the effect of lipid nanoparticles delivering mRNA at the animal level

[0094] Balb / C mice aged 6-8 weeks were randomly divided into 4 groups of 3 mice each and injected via the tail vein with PEG-LNP / mLuci, Chol-EK-LNP / mLuci, Ste-EK-LNP / mLuci, or Ole-EK-LNP / mLuci, with a dose of 5 μg of Luci-mRNA per mouse. Luciferase expression in vivo was observed by in vivo imaging 6 hours after injection, and fluorescence was quantified. Figure 7 As shown in the results, compared with the traditional PEG component, the in vivo transfection effect of LNPs containing zwitterionic peptide lipids was improved, among which the luciferase expression of Ste-EK8-LNP increased by 1.62 times, and the luciferase expression of Ole-EK8-LNP increased by 1.23 times, indicating that the replacement of the PEG lipid component of lipid nanoparticles by zwitterionic peptide lipids promoted the ability to deliver mRNA molecules into animals.

[0095] In summary, the present invention provides a zwitterionic polypeptide lipid molecule, which is an EK polypeptide modified with a fatty acid, a diglyceride derivative, or a cholesterol derivative. This lipid molecule can serve as a substitute for the polyethylene glycol component in lipid nanoparticles and can be combined with cationic lipids, phospholipids, and cholesterol to form lipid nanoparticles for delivering nucleic acid molecules into animals or cells. Experimental verification has shown that lipid nanoparticles composed of these zwitterionic polypeptide lipid molecules have uniform and stable particle size. Compared with PEG lipids, these lipid nanoparticles have improved cell transfection ability both in vivo and in vitro, without significant cytotoxicity. Therefore, these zwitterionic polypeptide lipid molecules can be used to prepare lipid nanoparticles with cationic lipids, phospholipids, and cholesterol to promote the in vivo or in vitro delivery of nucleic acid molecules.

Claims

1. A zwitterionic polypeptide lipid molecule, characterized in that: The structural formula of the zwitterionic polypeptide lipid molecule is any one of formulas (II) to (IV), 2. A method for preparing a zwitterionic polypeptide lipid molecule according to claim 1, characterized in that: include: First, the peptide (EK) was synthesized on a solid phase resin using solid phase synthesis method. n , n is 8, then stearic acid, oleic acid and glycerol dioleate succinate monoester are added to cap the polypeptide, and then a lysis solution is added to remove the solid phase resin and the side chain protecting groups to obtain the zwitterionic polypeptide lipid molecule.

3. Use of the zwitterionic polypeptide lipid molecule as claimed in claim 1 in preparing lipid nanoparticle carriers.

4. A lipid nanoparticle carrier, characterized in that The composition comprises cationic lipid molecules, phospholipids, cholesterol and the zwitterionic polypeptide lipid molecules as claimed in claim 1.

5. Use of the lipid nanoparticle carrier as claimed in claim 4 in the preparation of nucleic acid drugs.

6. The use according to claim 5, characterized in that The application includes: adding cationic lipid molecules, phospholipids, cholesterol and zwitterionic polypeptide lipid molecules into an acidic buffer containing nucleic acid, self-assembling to form lipid nanoparticles encapsulating nucleic acid, and preparing the nucleic acid drug.

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