Lipid-like molecule for selectively delivering nucleic acid medicine in organ, lipid-like nano-particle and application thereof, lipid-like nano-particle-nucleic acid compound and preparation method of lipid-like nano-particle-nucleic acid compound

Abstract

The invention relates to the technical field of biological medicines, in particular to a lipid molecule for selectively delivering nucleic acid drugs in organs, lipid nanoparticles and application thereof, a lipid nanoparticle-nucleic acid compound and a preparation method thereof. The lipoid molecule provided by the invention is obtained by carrying out Michael addition reaction on polyethyleneimine and aryl acrylate, the synthesis method is simple and safe, the reaction condition is mild, and the cost is low; the lipid nanoparticles are prepared from the following components in percentage by mass: 30 to 80 percent of lipid molecules, 5 to 50 percent of sterol, 5 to 40 percent of polyethylene glycol-lipid and 5 to 50 percent of auxiliary lipid. The method has the advantage that the organ selective delivery of the nucleic acid medicine by the lipid-like nano-particles in a tested organism is realized by adjusting the chemical structure of the lipid-like molecule.

Description

Technical Field

[0001] The present invention relates to the field of biopharmaceutical technology, and in particular to a lipid-like molecule, a lipid-like nanoparticle and its application for organ-selective delivery of nucleic acid drugs, and a lipid-like nanoparticle-nucleic acid complex and its preparation method. Background Art

[0002] As a new generation of biotechnological drugs, nucleic acids can regulate the expression of proteins at the gene level according to the central dogma of genetics, thereby achieving the purpose of disease treatment. Compared with traditional small molecule or antibody drugs, nucleic acid drugs have the advantages of high specificity, simple and efficient development, and wide treatment range, etc., and thus show great application potential in the prevention and treatment of various diseases. However, due to its large molecular weight, strong negative charge, easy to be degraded by enzymes and other characteristics, nucleic acid drugs face multiple barriers in actual application, resulting in low utilization rate. At present, developing safe and efficient carriers for delivering nucleic acid drugs is a favorable means to overcome the application barriers, especially the COVID-19 virus has further promoted the research and development of nucleic acid drug delivery carriers.

[0003] Non-viral carriers such as lipid / lipid-like nanoparticles (LNPs), polymer nanoparticles, and liposome polyplexes have received extensive attention in nucleic acid drug delivery. Among them, LNPs based on ionizable lipids / lipid-like molecules are the current most advanced nucleic acid drug delivery carriers, and a total of 3 nucleic acid drugs approved by the FDA for marketing adopt the LNP delivery technology. Traditional LNPs contain four components: ionizable lipids / lipid-like molecules, cholesterol, polyethylene glycol-lipid, and helper lipids. Among them, cholesterol can improve the stability and membrane fusion of LNPs; polyethylene glycol-lipid is used to regulate the particle size of LNPs and prevent aggregation to maintain the stability of its preparation; helper lipids are mainly used to assist in encapsulating nucleic acid molecules and enhance the stability of LNPs; ionizable lipids / lipid-like molecules, as the core components of LNPs, can effectively encapsulate nucleic acid molecules and promote their endosomal escape. Although LNPs have made significant progress as nucleic acid drug delivery carriers in clinical practice, there is still a problem that it is difficult to achieve organ-selective delivery after systemic administration. To solve this key problem, existing technologies mostly take measures such as adding additional auxiliary components on the basis of traditional four components or modifying targeted biomolecules on the surface of nanoparticles. However, these solutions have problems such as increased cost and process complexity in actual application. Therefore, designing and developing novel ionizable lipids / lipid-like molecules to achieve organ-selective nucleic acid drug delivery of LNPs by adjusting their own structures has important research value and broad application prospects. Summary of the Invention

[0004] In view of this, the object of the present invention is to provide a lipid-like molecule for organ-selective delivery of nucleic acid drugs, a lipid-like nanoparticle and its application, a lipid-like nanoparticle-nucleic acid complex and its preparation method. The lipid-like molecule provided by the present invention realizes the organ-selective delivery of nucleic acid drugs by its lipid-like nanoparticles in a test organism, and at the same time has good biosafety.

[0005] In order to achieve the above object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a lipid-like molecule obtained by Michael addition reaction of polyethyleneimine and aryl acrylate; the molecular weight of the polyethyleneimine is 0.3-1.2 kDa;

[0007] The aryl acrylate has a structure shown in any one of Formula I to Formula III:

[0008]

[0009] In Formula I to Formula III, n1 is an integer between 0 and 10, and n2 and n3 are respectively integers between 1 and 10; R1 to R3 are hydrogen atoms or hydrophobic lipid chains, and the hydrophobic lipid chains are selected from branched or unbranched, saturated or unsaturated, halogenated or unhalogenated aliphatic hydrocarbon groups with 1 to 20 carbon atoms.

[0010] Preferably, in Formula I to Formula III, n1 is an integer between 0 and 6, and n2 and n3 are respectively integers between 1 and 6; R1 to R3 are hydrogen atoms or hydrophobic lipid chains, and the hydrophobic lipid chains are selected from unbranched, saturated, unhalogenated aliphatic hydrocarbon groups with 1 to 5 carbon atoms.

[0011] Preferably, the polyethyleneimine is linear polyethyleneimine or branched polyethyleneimine.

[0012] The present invention provides a preparation method of the lipid-like molecule described in the above solution, including the following steps: performing a Michael addition reaction on polyethyleneimine and aryl acrylate to obtain the lipid-like molecule.

[0013] Preferably, the molar ratio of the aryl acrylate to the polyethyleneimine is 0.1-50:1; the temperature of the Michael addition reaction is 0-100 °C, and the reaction time is 4-72 h.

[0014] The present invention provides a lipid-like nanoparticle, which comprises the following components by mass percentage: 30-80% of the lipid-like molecule described in the above solution, 5-50% of sterol, 5-40% of polyethylene glycol-lipid, and 5-50% of auxiliary lipid.

[0015] The present invention provides the use of the lipid nanoparticles described in the above solution in the preparation of drugs for treating or preventing diseases. When the molecular weight of polyethyleneimine is 0.6 - 1.2 kDa, the disease is liver disease; when the molecular weight of polyethyleneimine ≥ 0.3 kDa and < 0.6 kDa, the disease is spleen disease.

[0016] The present invention provides a lipid nanoparticle-nucleic acid complex, comprising a nucleic acid drug and lipid nanoparticles encapsulating the nucleic acid drug; the lipid nanoparticles are the lipid nanoparticles described in the above solution.

[0017] Preferably, the nucleic acid drug is selected from one or more of aptamers, antisense oligonucleotides, small interfering RNAs, messenger RNAs, microRNAs, circular RNAs, clustered regularly interspaced short palindromic repeat (CRISPR)-associated nucleic acids, single guide RNAs, and plasmid DNAs.

[0018] The present invention provides a method for preparing the lipid nanoparticle-nucleic acid complex described in the above solution, comprising the following steps: dissolving lipid molecules, sterols, polyethylene glycol-lipids, and co-lipids in polar organic solvents respectively, and mixing them in a ratio to obtain an organic phase lipid premix;

[0019] dissolving the nucleic acid drug in a buffer solution to obtain an aqueous phase nucleic acid solution;

[0020] mixing the organic phase lipid premix with the aqueous phase nucleic acid solution to form a lipid nanoparticle-nucleic acid complex.

[0021] The present invention explores the influence of the molecular weight of polyethyleneimine (PEI) used in lipid molecules on the mRNA transfection effect of its lipid nanoparticles: within a specific molecular weight range (0.3 - 1.2 kDa), the smaller the molecular weight of PEI used in lipid molecules, the better the transfection effect of its lipid nanoparticle-mRNA complex on cells; when 0.3 kDa PEI is used in lipid molecules, its lipid nanoparticle-mRNA complex can achieve spleen-selective mRNA transfection in mice after intravenous injection, while when 0.6 kDa or 1.2 kDa PEI is used in lipid molecules, its lipid nanoparticle-mRNA complex will achieve liver-selective mRNA transfection in mice after intravenous injection. In other words, when the PEI molecular weight < 0.6 kDa, the obtained lipid nanoparticle-mRNA complex has spleen selectivity, and when the PEI molecular weight ≥ 0.6 kDa, the obtained lipid nanoparticle-mRNA complex has liver selectivity.

[0022] The lipid-like molecules provided by the present invention can achieve organ-selective nucleic acid drug delivery of their lipid-like nanoparticles in a test organism only by changing their own chemical structure, without the need to additionally add other components such as auxiliary lipids or targeting antibodies. The operation is simple and feasible and can effectively control costs.

[0023] The present invention provides a preparation method of the lipid-like molecules. The synthesis method is safe, simple, mild in conditions, low in raw material cost and easy to obtain.

[0024] The present invention provides a lipid-like nanoparticle, which effectively realizes spleen-selective nucleic acid delivery and helps to expand the application of non-viral delivery vectors in nucleic acid drug therapy. Description of the Drawings

[0025] Figure 1 1H NMR spectra of the lipid-like molecules obtained in Examples 1 to 3, where a is Example 1, b is Example 2, and c is Example 3;

[0026] Figure 2 Appearance diagram and Tyndall phenomenon diagram of the lipid-like nanoparticle-mRNA complex in Example 4; where a is the appearance diagram and b is the Tyndall phenomenon diagram;

[0027] Figure 3 Characterization diagrams of the physical and chemical properties of the lipid-like nanoparticle-mRNA complexes obtained in Examples 4 to 6, where a is the hydrated particle size diagram, b is the polydispersity coefficient diagram, c is the particle size distribution diagram, and d is the transmission electron microscopy diagram;

[0028] Figure 4 Diagram of the encapsulation efficiency of mRNA by the lipid-like nanoparticles in Examples 4 to 6;

[0029] Figure 5 Diagram of the transfection efficiency of the lipid-like nanoparticle-enhanced green fluorescent protein (EGFP) mRNA complex in HeLa cells in vitro in Application Example 1, where the co-incubation time of the complex and HeLa cells is 24 h; a is the fluorescence microscope photograph, and b, c, and d are all flow cytometry quantitative analysis diagrams;

[0030] Figure 6 Diagram for evaluating the cytotoxicity of the lipid-like nanoparticle-mRNA complex on HeLa cells at different concentrations in Application Example 2;

[0031] Figure 7 Ex vivo bioluminescence imaging pictures and bioluminescence quantitative analysis diagrams of the main organs (heart, liver, spleen, lung, kidney) of mice after the lipid-like nanoparticle-luciferase (Luc) mRNA complex is intravenously injected into the mice for 24 h in Application Example 3;

[0032] Figure 8Pathological evaluation diagrams of the main organs (heart, liver, spleen, lung, kidney) of mice after intravenous injection of lipid nanoparticle - mRNA complexes at an mRNA concentration that enables effective transfection in Application Example 4. Detailed implementation mode

[0033] The present invention provides a lipid molecule obtained by Michael addition reaction of polyethyleneimine and aryl acrylate.

[0034] In the present invention, the polyethyleneimine can be linear polyethyleneimine or branched polyethyleneimine. In the present invention, the molecular weight of the polyethyleneimine (PEI) is 0.3 - 1.2 kDa, and in specific embodiments, it can be 0.3 kDa, 0.6 kDa or 1.2 kDa. In the present invention, the molecular weight of PEI affects the targeting of its lipid nanoparticles. When the molecular weight of PEI is 0.3 - 1.2 kDa, its lipid nanoparticles can achieve organ - selective nucleic acid drug delivery in the test organisms.

[0035] In the present invention, the aryl acrylate has the structure shown in any one of Formula Ⅰ - Ⅲ:

[0036]

[0037]

[0038] In Formula Ⅰ - Ⅲ, n1 is an integer between 0 and 10, preferably 0 - 6; n2 and n3 are respectively integers between 1 and 10, preferably 1 - 6; R1 - R3 are hydrogen atoms or hydrophobic lipid chains, and the hydrophobic lipid chains are selected from branched or unbranched, saturated or unsaturated, halogenated or unhalogenated aliphatic hydrocarbon groups with 1 - 20 carbon atoms; more preferably, the hydrophobic lipid chains are selected from unbranched, saturated, unhalogenated aliphatic hydrocarbon groups with 1 - 5 carbon atoms; further preferably, the aryl acrylate is selected from phenyl acrylate, benzyl acrylate, 2 - phenylethyl acrylate, 2 - phenoxyethyl acrylate, phenoxy polyethylene glycol acrylate or 4 - ethylphenyl acrylate.

[0039] The present invention provides a preparation method of the lipid molecule described in the above - mentioned scheme, including the following steps: performing Michael addition reaction on polyethyleneimine and aryl acrylate to obtain the lipid molecule.

[0040] In the present invention, the molar ratio of the aryl acrylate to polyethyleneimine is preferably 0.1 - 50:1, more preferably 1 - 30:1, and in specific embodiments, it can be 5:1, 6:1, 7:1, 10:1, 12:1, 14:1, 20:1, 24:1 or 28:1.

[0041] In the present invention, the temperature of the Michael addition reaction is preferably 0 to 100 °C, more preferably 0 to 50 °C, and in specific embodiments, it can be 0 °C, 10 °C, 20 °C, 25 °C, 30 °C, 40 °C or 50 °C; the time of the Michael addition reaction is preferably 4 to 72 h, more preferably 4 to 36 h, and in specific embodiments, it can be 4 h, 10 h, 20 h, 24 h, 30 h or 36 h. In the present invention, the Michael addition reaction is preferably carried out under light-shielding and stirring conditions.

[0042] After completing the Michael addition reaction, in the present invention, the crude product obtained by synthesis is preferably washed with absolute ethanol and centrifuged to precipitate, and then dried to obtain lipid-like molecules.

[0043] The present invention provides a lipid-like nanoparticle, which comprises the following components by mass percentage: 30 to 80% of the lipid-like molecules described in the above scheme, 5 to 50% of sterols, 5 to 40% of polyethylene glycol-lipids, and 5 to 50% of auxiliary lipids.

[0044] By mass percentage, the lipid-like nanoparticle provided by the present invention comprises 30 to 80% of lipid-like molecules, and in specific embodiments, it can be 30%, 40%, 50%, 60%, 70% or 80%.

[0045] By mass percentage, the lipid-like nanoparticle provided by the present invention comprises 5 to 50% of sterols, and in specific embodiments, it can be 5%, 10%, 20%, 30%, 40% or 50%. In the present invention, the sterol is preferably one or more of animal sterols, plant sterols and fungal sterols, the animal sterol can be cholesterol; the plant sterol can be one or more of β-sitosterol, sitostanol, stigmasterol, stigmastanol, campesterol and ergosterol; the fungal sterol can be ergosterol.

[0046] By mass percentage, the lipid-like nanoparticle provided by the present invention comprises 5 to 40% of polyethylene glycol-lipids, and in specific embodiments, it can be 5%, 10%, 20%, 30% or 40%. In the present invention, the polyethylene glycol-lipid can be one of 1,2-dimyristoyl-rac-glycerol-3-methoxy-polyethylene glycol 2000 (DMG-PEG), distearoyl phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG), d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS), 2-myrstoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol 2000 (DMPE-PEG), L-phosphatidylethanolamine-polyethylene glycol 2000 (DPPE-PEG), 1,2-dipalmitoyl-sn-glycero-polyethylene glycol 2000 (DPG-PEG) and 1,2-distearoyl-sn-glycero-polyethylene glycol 2000 (DSG-PEG).

[0047] In terms of mass percentage, the lipid nanoparticles provided by the present invention include 5-50% of co-lipid, which can be 5%, 10%, 20%, 30%, 40% or 50% in specific embodiments. In the present invention, the co-lipid can be one of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), 1,2-dimyristoyl-sn-glycero-3-phosphate-rac-(1-glycerol) (DPPG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dipalmitoyl-rac-glycerol-3-phosphocholine (DPPC) and dimyristoyl phosphatidylcholine (DMPC).

[0048] In the present invention, the hydrated particle size of the lipid nanoparticles is preferably ≤150 nm, more preferably 50-150 nm.

[0049] The present invention provides the application of the lipid nanoparticles described in the above scheme in the preparation of drugs for treating or preventing diseases. When the molecular weight of polyethyleneimine is 0.6-1.2 kDa, the disease is liver disease; when the molecular weight of polyethyleneimine ≥0.3 kDa and <0.6 kDa, the disease is spleen disease. The present invention does not make special limitations on the specific types of the liver disease and spleen disease; specifically, liver diseases such as hepatitis and liver cancer; spleen diseases such as splenic lymphoma and splenic metastatic tumor can also be used to prevent or treat spleen immune function-related diseases such as melanoma and rheumatoid arthritis.

[0050] The present invention provides a lipid nanoparticle-nucleic acid complex, which includes a nucleic acid drug and lipid nanoparticles encapsulating the nucleic acid drug; the lipid nanoparticles are the lipid nanoparticles described in the above scheme.

[0051] In the present invention, the nucleic acid drug can be selected from one or more of nucleic acid aptamer (Aptamer), antisense oligonucleotide (ASO), small interfering RNA (siRNA), messenger RNA (mRNA), microRNA (microRNA), circular RNA (circRNA), clustered regularly interspaced short palindromic repeats (GRISPR)-related nucleic acid, single guide RNA (sgRNA) and plasmid DNA (pDNA).

[0052] The present invention has no special requirements for the encapsulation efficiency of the nucleic acid drug, which can be adjusted according to actual needs. In the embodiments of the present invention, the encapsulation efficiency of the nucleic acid drug exceeds 85%.

[0053] The present invention provides a method for preparing the lipid nanoparticle-nucleic acid complex described in the above solution, comprising the following steps: dissolving lipid molecules, sterols, polyethylene glycol-lipids, and co-lipids in polar organic solvents respectively, and mixing them in proportion to obtain an organic phase lipid premix;

[0054] Dissolving the nucleic acid drug in a buffer solution to obtain an aqueous phase nucleic acid solution;

[0055] Mixing the organic phase lipid premix with the aqueous phase nucleic acid solution to form a lipid nanoparticle-nucleic acid complex.

[0056] In the present invention, lipid molecules, sterols, polyethylene glycol-lipids, and co-lipids are dissolved in organic solvents respectively, and mixed in proportion to obtain an organic phase lipid premix.

[0057] In the present invention, the polar organic solvent is preferably one or more of absolute ethanol, methanol, tert-butanol, tetrahydrofuran, dimethyl sulfoxide, and N,N-dimethylformamide; the amount of the polar organic solvent used preferably satisfies that the solute can be completely dissolved.

[0058] In the present invention, the nucleic acid drug is dissolved in a buffer solution to obtain an aqueous phase nucleic acid solution.

[0059] In the present invention, the buffer solution is preferably an acetic acid-sodium acetate buffer solution or a citric acid-sodium citrate buffer solution; the pH value of the buffer solution is preferably 3.0-8.0, and can be 3.0, 4.0, 5.0, 6.0, 7.0, or 8.0 in specific embodiments. The present invention has no special requirements for the amount of the buffer solution, as long as the nucleic acid drug can be completely dissolved.

[0060] After obtaining the organic phase lipid premix and the aqueous phase nucleic acid solution, the present invention mixes the organic phase lipid premix with the aqueous phase nucleic acid solution to form a lipid nanoparticle-nucleic acid complex.

[0061] In the present invention, the mass ratio of lipid molecules in the organic phase lipid premix to the nucleic acid drug in the aqueous phase nucleic acid solution is preferably 1-40:1, and can be 1:1, 5:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, or 40:1 in specific embodiments.

[0062] In the present invention, the mixing is preferably carried out in a microfluidic device; during the mixing process, the two-phase solutes self-assemble through electrostatic interaction, hydrophilic-hydrophobic interaction, and van der Waals force interaction to obtain a lipid nanoparticle-nucleic acid complex encapsulating the nucleic acid drug, and the solution state changes from a transparent true solution to a white colloidal solution.

[0063] After the above mixing, the obtained lipid nanoparticles-nucleic acid complex is dispersed in a polar organic solvent. Preferably, the obtained system is dialyzed in 1×PBS buffer (pH 7.4) to remove the polar organic solvent, and a lipid nanoparticles-nucleic acid complex that can be used in biological experiments is obtained.

[0064] The following examples are used to illustrate in detail the lipid molecules, lipid nanoparticles and their applications for organ-selective delivery of nucleic acid drugs, lipid nanoparticles-nucleic acid complexes and their preparation methods provided by the present invention, but they should not be construed as limiting the protection scope of the present invention.

[0065] Example 1

[0066] A method for synthesizing a lipid molecule is as follows:

[0067] 2-Phenylethyl acrylate and branched PEI with a molecular weight of 0.3 kDa (polyethyleneimine M.W. 300, CAS number 9002-98-6, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) were added to a reaction flask at a molar ratio of 6:1 and mixed evenly. The mixture was continuously magnetically stirred at 25 °C in the dark for 36 h. The crude product obtained from the synthesis was washed with absolute ethanol and centrifuged to precipitate, and after drying, a lipid molecule was obtained, which was named 6:1 according to the molar ratio of the feed. The obtained lipid molecule was subjected to 1H NMR analysis, and the obtained 1H NMR spectrum is as shown in Figure 1 a in the figure, which proves that the target product was obtained. Note: For the convenience of observation, the 1H NMR spectrum was partially enlarged, resulting in incomplete display of the solvent peak in the height direction. This is hereby explained.

[0068] Example 2

[0069] A method for synthesizing a lipid molecule is as follows:

[0070] 2-Phenylethyl acrylate and branched PEI with a molecular weight of 0.6 kDa (polyethyleneimine M.W. 600, CAS number 9002-98-6, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) were added to a reaction flask at a molar ratio of 12:1 and mixed evenly. The mixture was continuously magnetically stirred at 25 °C in the dark for 36 h. The crude product obtained from the synthesis was washed with absolute ethanol and centrifuged to precipitate, and after drying, a lipid molecule was obtained, which was named 12:1 according to the molar ratio of the feed. The obtained lipid molecule was subjected to 1H NMR analysis, and the obtained 1H NMR spectrum is as shown in Figure 1 b in the figure, which proves that the target product was obtained.

[0071] Example 3

[0072] A method for synthesizing a lipid molecule is as follows:

[0073] 2-Phenylethyl acrylate and branched PEI with a molecular weight of 1.2 kDa (polyethyleneimine M.W. 1200, CAS No. 9002-98-6, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) were respectively added to a reaction flask at a molar ratio of 24:1 and mixed evenly. Under the conditions of 25 °C and in the dark, magnetic stirring was continued for 36 h. The obtained crude product was washed with absolute ethanol and centrifuged to precipitate, and after drying, a lipid-like molecule was obtained, which was named 24:1 according to the feeding molar ratio. The obtained lipid-like molecule was subjected to 1H NMR analysis, and the obtained 1H NMR spectrum was as shown in Figure 1 c in the figure, proving that the target product was obtained.

[0074] Examples 4-6

[0075] A method for preparing lipid nanoparticles encapsulating mRNA is as follows:

[0076] The lipid-like molecules obtained in Examples 1-3 were respectively mixed with cholesterol, DOPE, and DMG-PEG2000 according to a mass ratio of 50%, 20%, 20%, and 10%, and dissolved in an absolute ethanol solution to obtain an organic phase lipid premix. mRNA (EZ Cap TM EGFP mRNA (5-moUTP), purchased from APExBIO Technology Co., Ltd., USA) was dissolved in a citric acid-sodium citrate buffer solution with a concentration of 10 mM (pH 4.0) to obtain an aqueous phase mRNA solution. Using a microfluidic device, the organic phase lipid premix and the aqueous phase mRNA solution were uniformly mixed at a volume ratio of 1:3, where the mass ratio of the lipid-like molecule to mRNA was 10:1, to obtain lipid nanoparticles encapsulating mRNA. The obtained lipid nanoparticles encapsulating mRNA were dialyzed in 1×PBS buffer (pH 7.4) to remove absolute ethanol, and a lipid nanoparticle-mRNA complex available for biological experiments was obtained (as shown in Figure 2 shown, Figure 2 a in the figure is a photo of the appearance without laser light source irradiation, and b is a photo of the Tyndall phenomenon under red laser light source irradiation), and they were respectively named 6:1 (Example 4), 12:1 (Example 5), and 24:1 (Example 6).

[0077] Dynamic light scattering (DLS) and transmission electron microscopy (TEM) were used to characterize the particle size and morphology of the obtained lipid nanoparticle-mRNA complex. As shown in Figure 3 shown, the obtained lipid nanoparticle-mRNA complex is a spherical structure (see Figure 3 d in the figure), the hydrated particle size is less than 150 nm (see Figure 3 a in the figure) and the polydispersity index (PDI) is less than 0.3, that is, the particle size distribution is relatively uniform ( Figure 3In b and c). The encapsulation efficiency of lipid nanoparticles on mRNA in the obtained lipid nanoparticle-mRNA complexes was determined using a Ribogreen kit. As Figure 4 shown, the encapsulation efficiency of lipid nanoparticles on mRNA exceeded 85%. Specifically, the encapsulation rates of the three types of nanoparticles, 6:1, 12:1, and 24:1, on mRNA were 88.1 ± 2.2%, 87.2 ± 3.7%, and 87.3 ± 2.4%, respectively.

[0078] Application Example 1

[0079] The lipid nanoparticle-mRNA complexes obtained in Examples 4 to 6 were incubated with HeLa cells at an mRNA concentration of 1 μg / mL, where the mRNA was mRNA encoding enhanced green fluorescent protein (EGFP). After incubation in a cell culture incubator at 37°C and 5% CO2 for 24 h, the expression effect of EGFP in HeLa cells was observed and recorded using a fluorescence microscope ( Figure 5 in a), and the fluorescence intensity and protein expression efficiency in the cells were quantitatively analyzed using a flow cytometer ( Figure 5 in b, c, and d). The experimental results showed that the three types of lipid nanoparticle-mRNA complexes, 6:1, 12:1, and 24:1, all exhibited excellent transfection efficiency, and more than 99% of the cells successfully expressed green fluorescent protein.

[0080] Application Example 2

[0081] The lipid nanoparticle-mRNA complex named 6:1 obtained in Example 4 was incubated with HeLa cells at mRNA concentrations of 0.5 μg / mL, 1 μg / mL, 2 μg / mL, and 4 μg / mL, where the mRNA was mRNA encoding enhanced green fluorescent protein (EGFP). After incubation in a cell culture incubator at 37°C and 5% CO2 for 24 h, the cell viability of HeLa cells was detected using a CCK-8 kit. As Figure 6 shown, under the four mRNA concentration conditions, the cell survival rate was above 95%, indicating that the lipid nanoparticles had good biosafety performance.

[0082] Application Example 3

[0083] The lipid nanoparticle - mRNA complexes obtained in Examples 4 - 6 were intravenously injected into mice at an mRNA dose of 0.4 μg / g, where the mRNA was the mRNA of luciferase (Luc). 24 hours after intravenous injection, the mice were intraperitoneally injected with a luciferin substrate. After 10 minutes, a small animal in - vivo imaging system was used to observe and record the bioluminescence signals in the major organs (heart, liver, spleen, lung, kidney) of the mice, and the bioluminescence signals were quantitatively analyzed to evaluate the transfection efficiency of the lipid nanoparticle - mRNA complexes in various organs of the mice. As Figure 7 shown, the three lipid nanoparticle - mRNA complexes with ratios of 6:1, 12:1, and 24:1 all showed effective mRNA transfection in mice. Among them, the 6:1 lipid nanoparticle - mRNA complex mainly achieved mRNA transfection in the spleen, while the 12:1 and 24:1 lipid nanoparticle - mRNA complexes mainly achieved mRNA transfection in the liver.

[0084] Application Example 4

[0085] The lipid nanoparticle - mRNA complex named 6:1 obtained in Example 4 was intravenously injected into mice at an mRNA dose of 0.4 μg / g. 24 hours later, the major organs (heart, liver, spleen, lung, kidney) of the mice were stained with H&E for pathological examination. As Figure 8 shown, compared with the organs of untreated mice, no obvious lesions were observed in the organs of mice treated with the above - mentioned complex, indicating that the lipid nanoparticle - mRNA complex provided by the present invention has good biocompatibility in the test organisms at a dose that can achieve effective transfection.

[0086] The above - mentioned are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A lipid molecule, characterized in that, It is obtained by the Michael addition reaction of polyethyleneimine and aryl acrylate; the molecular weight of the polyethyleneimine is 0.3 to 1.2 kDa; The aryl acrylate has the structure shown in any one of Formula I to Formula III: In Formula I to Formula III, n1 is an integer between 0 and 10, and n2 and n3 are respectively integers between 1 and 10; R1 to R3 are hydrogen atoms or hydrophobic lipid chains, and the hydrophobic lipid chains are selected from branched or unbranched, saturated or unsaturated, halogenated or unhalogenated aliphatic hydrocarbon groups with 1 to 20 carbon atoms.

2. The lipid molecule according to claim 1, wherein In the above Formula I to Formula III, n1 is an integer between 0 and 6, and n2 and n3 are respectively integers between 1 and 6; R1 to R3 are hydrogen atoms or hydrophobic lipid chains, and the hydrophobic lipid chains are selected from unbranched, saturated, unhalogenated aliphatic hydrocarbon groups with 1 to 5 carbon atoms.

3. The lipid molecule according to claim 1, wherein, The polyethyleneimine is linear polyethyleneimine or branched polyethyleneimine.

4. The method for preparing the lipid molecule according to any one of claims 1 to 3, comprising the following steps: The polyethyleneimine and aryl acrylate are subjected to a Michael addition reaction to obtain the lipid-like molecule.

5. The preparation method according to claim 4, characterized in that, The molar ratio of the aryl acrylate to the polyethyleneimine is 0.1 to 50:1; the temperature of the Michael addition reaction is 0 to 100 °C, and the reaction time is 4 to 72 h.

6. A lipid nanoparticle, characterized in that, By mass percentage, it includes the following components: 30 to 80% of the lipid-like molecule according to any one of claims 1 to 3, 5 to 50% of sterol, 5 to 40% of polyethylene glycol-lipid, and 5 to 50% of co-lipid.

7. Use of the lipid nanoparticles according to claim 6 in preparing a drug for treating or preventing a disease, characterized in that: When the molecular weight of the polyethyleneimine is 0.6 to 1.2 kDa, the disease is liver disease; when the molecular weight of the polyethyleneimine is ≥0.3 kDa and <0.6 kDa, the disease is spleen disease.

8. A lipid nanoparticle-nucleic acid complex, characterized in that: It includes a nucleic acid drug and lipid-like nanoparticles encapsulating the nucleic acid drug; the lipid-like nanoparticles are the lipid-like nanoparticles according to claim 6.

9. The lipid nanoparticle-nucleic acid complex according to claim 8, wherein, The nucleic acid drug is selected from one or more of nucleic acid aptamers, antisense oligonucleotides, small interfering RNAs, messenger RNAs, microRNAs, circular RNAs, clustered regularly interspaced short palindromic repeat sequence-related nucleic acids, single guide RNAs, and plasmid DNAs.

10. The method for preparing the lipid nanoparticle-nucleic acid complex according to claim 8 or 9, comprising the following steps: The lipid-like molecule, sterol, polyethylene glycol-lipid, and co-lipid are respectively dissolved in a polar organic solvent and mixed in proportion to obtain an organic phase lipid premix; The nucleic acid drug is dissolved in a buffer solution to obtain an aqueous phase nucleic acid solution; The organic phase lipid premix is mixed with the aqueous phase nucleic acid solution to form a lipid-like nanoparticle-nucleic acid complex.

Images