Ionizable lipid based on tris (2-aminoethyl) amine, lipid nanoparticles and preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and relates to tri (2-aminoethyl) amine-based ionizable lipid, lipid nanoparticles and a preparation method and application thereof. The chemical structure of the compound is shown as a formula I. In the formula I, R1 and R2 are respectively and independently selected from substituted or unsubstituted C8-C14 alkyl, substituted or unsubstituted C8-C14 alkenyl and substituted or unsubstituted C8-C14 alkynyl. The lipid nanoparticles formed by the ionizable lipid based on the tris (2-aminoethyl) amine, provided by the invention, have the advantages of biodegradability and high transfection efficiency.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to an ionizable lipid based on tris(2-aminoethyl)amine, lipid nanoparticles, their preparation methods and applications. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Gene therapy has become a research hotspot for the treatment of various diseases. Nucleic acid drugs, which are exogenous DNA or RNA drugs with specific base sequences, have advantages such as high designability, high specificity, short development cycle, low likelihood of drug resistance, and great therapeutic potential. However, due to the difficulty of nucleic acid molecules penetrating cell membranes and their rapid clearance by blood circulation, effectively delivering nucleic acid drugs to target sites remains a challenge. Therefore, developing safe and efficient nucleic acid delivery vectors is key to realizing the application potential of gene therapy.

[0004] Existing technologies utilize lipid nanoparticles (LNPs) to maximize the delivery of exogenous nucleic acid drugs to target cells while protecting the nucleic acids from degradation by nucleases in plasma and tissues. LNPs typically consist of ionizable lipids, auxiliary lipids, cholesterol, and polyethylene glycol-modified lipids (PEG-lipids). The ionizable lipids are the core component, exhibiting neutrality under physiological conditions (pH 7.4) to reduce toxicity, while protonation in acidic endosomes promotes escape. To date, only a few nucleic acid payloads have been able to escape from late endosomes into the cytoplasm via LNPs, resulting in low endosome escape efficiency and consequently low transfection efficiency. Furthermore, most ionizable lipid compounds are synthesized via epoxide ring-opening or Michael addition reactions, exhibiting poor biodegradability. Therefore, developing novel ionizable lipids with efficient endosome escape capabilities and good biodegradability is crucial for the innovation and application of nucleic acid drug delivery systems. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide an ionizable lipid based on tris(2-aminoethyl)amine, lipid nanoparticles, their preparation method, and applications. The lipid nanoparticles formed from the ionizable lipid based on tris(2-aminoethyl)amine provided by the present invention have the advantages of biodegradability and high transfection efficiency.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: Firstly, an ionizable lipid based on tris(2-aminoethyl)amine, the chemical structure of which is shown in Formula I,

[0007] R1 and R2 are independently selected from substituted or unsubstituted C8-C14 alkyl, substituted or unsubstituted C8-C14 alkenyl, and substituted or unsubstituted C8-C14 alkynyl, respectively.

[0008] On the other hand, a method for preparing an ionizable lipid based on tris(2-aminoethyl)amine includes the following reaction steps: An intermediate was obtained by Michael addition reaction of an acrylate derivative with γ-aminobutyric acid. The intermediate was subjected to an amidation reaction with tris(2-aminoethyl)amine to obtain the compound shown in Formula I; The chemical structural formula of the acrylate derivative is as follows: ; The chemical structural formula of the intermediate is ; R is R1 and / or R2, which are independently selected from substituted or unsubstituted C8-C14 alkyl, substituted or unsubstituted C8-C14 alkenyl, and substituted or unsubstituted C8-C14 alkynyl, respectively.

[0009] Thirdly, a lipid nanoparticle is composed of the tri(2-aminoethyl)amine-based ionizable lipid, auxiliary lipid, sterol, and PEG lipid described in the first aspect of the present invention.

[0010] Fourthly, the application of the lipid nanoparticles described in the third aspect of the present invention in the preparation of drug delivery carriers.

[0011] Fifthly, a pharmaceutical composition comprising an active ingredient and the lipid nanoparticles described in the third aspect of the present invention.

[0012] The beneficial effects of this invention are as follows: 1. The ionizable lipid provided by this invention uses γ-aminobutyric acid as a linker and amide bonds as connecting bonds. It has a dual degradable structure of amide and ester bonds, which can be rapidly hydrolyzed by enzymes in vivo, reducing its accumulation in vivo. It has good biodegradability and avoids long-term toxicity and immunogenicity.

[0013] 2. The ionizable lipids provided by this invention are neutral under physiological conditions (pH 7.4) and positively charged under acidic conditions. The ionizable lipids undergo protonation with changes in endosome pH, resulting in positively charged lipid compounds that form an unstable inverted hexagonal phase with the negatively charged endosome phospholipids. This disrupts endosome membrane stability and, combined with the hydrophobic tail, promotes the fusion of LNPs with the endosome membrane, facilitating efficient endosome escape of nucleic acid molecules and improving transfection efficiency.

[0014] 3. The ionizable lipid raw materials provided by this invention have low cost, simple synthesis steps, convenient product separation, and are easy to store.

[0015] 4. The ionizable lipids provided in this invention, along with total auxiliary lipids (phospholipids + fatty acid active substance docosahexaenoic acid), cholesterol, and PEG lipids, are used to prepare phagocytic lipid nanoparticles (DHA-based LNPs, DLNPs). This invention breaks through the traditional four-component limitation and endows LNPs with the basic function of enhancing cellular phagocytosis. Furthermore, it is uniform, stable, and highly safe, exhibiting superior performance in nucleic acid delivery systems, which is beneficial for the development and application of nucleic acid drugs. Attached Figure Description

[0016] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0017] Figure 1 This is a TEM image of DLNPs prepared from ionizable lipids in an embodiment of the present invention; Figure 2 The changes in particle size, PDI, encapsulation efficiency, and transfection efficiency of DLNPs prepared from ionizable lipids in this embodiment of the invention within one week; Figure 3 This is a potential characterization diagram of DLNPs prepared from ionizable lipids in the embodiments of the present invention. Detailed Implementation

[0018] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0019] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0020] The term "nucleic acid" in this invention refers to polymers of deoxyribonucleotides (DNA), ribonucleotides (RNA), and modified forms thereof, said polymers being in the form of single fragments or components of larger constructs, straight-chain or branched, single-stranded, double-stranded, triple-stranded, or hybrids thereof. The term also includes RNA / DNA hybrids.

[0021] The term "lipid" in this invention refers to a group of organic compounds, including but not limited to esters of fatty acids, and is generally characterized as being poorly soluble in water but soluble in many organic solvents.

[0022] The term "lipid nanoparticle" in this invention refers to a particle having a size on the nanometer scale that contains at least one lipid.

[0023] In this invention, "alkyl" refers to a saturated aliphatic hydrocarbon group, including straight-chain and branched alkyl groups. The substituents of C8-C14 alkyl groups are one or more halogens, hydroxyl groups, amino groups, carboxyl groups, nitro groups, amide groups, alkoxycarbonyl groups, alkylamide groups, dialkylamide groups, alkylamino groups, dialkylamino groups, thioalkyl groups, and heteroatom substituents (oxo, thio).

[0024] In this invention, the term "alkenyl" refers to an aliphatic hydrocarbon group containing at least one unsaturated carbon-carbon double bond, including straight-chain and branched alkenyl groups. The substituents in C8-C14 alkenyl groups are one or more selected from halogens, hydroxyl groups, amino groups, nitro groups, carboxyl groups, amide groups, alkoxycarbonyl groups, alkylamide groups, dialkylamide groups, alkylamino groups, dialkylamino groups, thioalkyl groups, and heteroatom substituents (oxo, thio).

[0025] In this invention, the term "alkynyl" refers to an aliphatic hydrocarbon group containing at least one unsaturated carbon-carbon triple bond, including straight-chain and branched alkynyl groups. The substituents of the C8-C14 alkynyl group are one or more selected from halogens, hydroxyl groups, amino groups, nitro groups, carboxyl groups, amide groups, alkoxycarbonyl groups, alkylamide groups, dialkylamide groups, alkylamino groups, dialkylamino groups, thioalkyl groups, and heteroatom substituents (oxo- or thio-).

[0026] In this invention, the term "substituted" refers to one or more hydrogen atoms in a group being substituted by a corresponding number of substituent groups independently of each other.

[0027] In order to develop an efficient and biodegradable nucleic acid drug delivery system, this invention proposes an ionizable lipid based on tris(2-aminoethyl)amine, lipid nanoparticles, their preparation method and application.

[0028] A typical embodiment of the present invention provides an ionizable lipid based on tris(2-aminoethyl)amine, the chemical structure of which is shown in Formula I.

[0029] R1 and R2 are independently selected from substituted or unsubstituted C8-C14 alkyl, substituted or unsubstituted C8-C14 alkenyl, and substituted or unsubstituted C8-C14 alkynyl, respectively.

[0030] In some embodiments, R1 and R2 are each independently selected from C8-C14 straight-chain alkyl groups.

[0031] In some embodiments, the compounds are selected from the following: (ZGB8) (ZGB10) (ZGB12) (ZGB14).

[0032] Another embodiment of the present invention provides a method for preparing an ionizable lipid based on tris(2-aminoethyl)amine, comprising the following reaction steps: An intermediate was obtained by Michael addition reaction of an acrylate derivative with γ-aminobutyric acid. The intermediate was subjected to an amidation reaction with tris(2-aminoethyl)amine to obtain the compound shown in Formula I; The chemical structural formula of the acrylate derivative is as follows: ; The chemical structural formula of the intermediate is ; R is R1 and / or R2, which are independently selected from substituted or unsubstituted C8-C14 alkyl, substituted or unsubstituted C8-C14 alkenyl, and substituted or unsubstituted C8-C14 alkynyl, respectively.

[0033] In some embodiments, R1 and R2 are each independently selected from C8-C14 straight-chain alkyl groups.

[0034] In some embodiments, R1 and R2 are C8 straight-chain alkyl, C10 straight-chain alkyl, C12 straight-chain alkyl, or C14 straight-chain alkyl.

[0035] In some embodiments, the Michael addition reaction is carried out at a temperature of 85–95 °C. Specifically, the reaction time is 22–26 h.

[0036] In some embodiments, the molar ratio of acrylate derivative to γ-aminobutyric acid is 1:(2.5~3.5).

[0037] In some embodiments, the carboxyl group of the intermediate is activated by EDCI (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) and HOBT (1-hydroxybenzotriazole) in the amidation reaction.

[0038] In some embodiments, the molar ratio of the intermediate to tris(2-aminoethyl)amine is (3.5~4.5):1.

[0039] In the preparation of ionizable lipids, the solvents used in this invention include, but are not limited to, methanol, ethanol, isopropanol, benzene, toluene, xylene, pentane, hexane, octane, cyclohexanediamine, cyclohexanone, methylcyclohexanone, chlorobenzene, dichlorobenzene, dichloromethane, diethyl ether, propylene oxide, acetone, methyl butyl ketone, methyl isobutyl ketone, N,N-dimethylformamide, N,N-dimethylacetamide, acetonitrile, pyridine, phenol, styrene, and triethanolamine. Specifically, isopropanol is used as the solvent for the Michael addition reaction. Specifically, dichloromethane is used as the solvent for the amidation reaction.

[0040] A third embodiment of the present invention provides a lipid nanoparticle composed of the above-mentioned ionizable lipid based on tris(2-aminoethyl)amine, auxiliary lipid, sterol and PEG lipid.

[0041] In some embodiments, the product also includes a fatty acid bioactive substance. Specifically, the fatty acid bioactive substance is docosahexaenoic acid (DHA). DHA can participate in the metabolism of polyunsaturated phospholipids in cell membranes, improve membrane fluidity, and enhance phagocytic function. Lipid nanoparticles are prepared by adding DHA to the traditional four-component lipids of LNPs to prepare phagocytogenic LNPs (DHA-based LNPs, DLNPs), endowing LNPs with the basic function of enhancing phagocytic ability. Specifically, the mass ratio of ionizable lipids to fatty acid bioactive substance is (15~45):(2.5~30).

[0042] In some embodiments, the sterol includes, but is not limited to, one or any combination of cholesterol, 20α-hydroxycholesterol, and β-sitosterol. Specifically, the sterol is cholesterol. Specifically, the molar ratio of ionizable lipid to sterol is (15~45):(20~35).

[0043] In some embodiments, the auxiliary phospholipid is selected from distearylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dioleoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoylphosphatidylethanolamine 4-(N-maleimidemethyl)-cyclohexanediamine-1-carboxylic acid ester (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearylphosphatidyl-phosphatidyl-ethanolamine (DSPE), monomethylphosphatidyl-phosphatidyl-phosphatidyl-ethanolamine, etc. Ethanolamine (e.g., 16-O-monomethyl PE), dimethyl phosphatidylethanolamine (e.g., 16-O-dimethyl PE), 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), hydrogenated soybean phosphatidylcholine (HSPC), lecithin choline (EPC), dioleoylphosphatidylserine (DOPS), sphingomyelin (SM), dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG), distearate phosphatidylglycerol (DSPG), disinoylphosphatidylcholine (DEPC), palmitoyloleoylphosphatidylglycerol (POPG), ditrans-oleoyl-phosphatidylethanolamine (DEPE), 1,2-dilauroyl-sn-glycerol-3-phosphate ethanolamine (DL) The phospholipid is selected from one or a mixture thereof, including 1,2-diphydanyl-sn-glycerol-3-phosphate ethanolamine (DPHyPE), lecithin, phosphatidylethanolamine, lysophosphatidylcholine, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, lecithin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, hexadecyl phosphate, lysophosphatidylcholine, and dilinoleylphosphatidylcholine. Specifically, the accessory phospholipid is DOPE. Specifically, the molar ratio of ionizable lipid to accessory phospholipid is (15~45):(10~40).

[0044] In some embodiments, PEG lipids include, but are not limited to, polyethylene glycol (PEG), 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG or DMG-PEG), distearate phosphatidylethanolamine-polyethylene glycol (DSPE-PEG), dipalmitoylphosphatidylethanolamine-methoxy-polyethylene glycol (DPPE-PEG), dimethacrylate-polyethylene glycol (PEG-DMA), 1,2-distearate-oxypropyl-3-amine-N-[methoxy(polyethylene glycol)] (PEG-DSA), methoxy-polyethylene glycol-phosphatidylethanolamine derivative mPEG-PE, methoxy-polyethylene glycol-ceramide derivative mPEG-Ceramide, functional PEG-liposomes, fluorescein-PEG-PE, and other derivatives. Specifically, the PEG lipid is DSPE-PEG2000. Specifically, the molar ratio of ionizable lipid to PEG lipid is (15~45):(0.5~5).

[0045] A fourth embodiment of the present invention provides an application of the above-mentioned lipid nanoparticles in the preparation of drug delivery carriers.

[0046] A fifth embodiment of the present invention provides a pharmaceutical composition comprising an active ingredient and the above-described lipid nanoparticles.

[0047] In some embodiments, the active ingredient is a drug for treating neurodegenerative diseases (such as Alzheimer's disease), cancer, autoimmune diseases, inflammation, or genetic diseases.

[0048] In some embodiments, the active ingredient is a nucleic acid drug. Specifically, the nucleic acid drug includes, but is not limited to, one or a combination of several of the following: siRNA, mRNA, microRNA, circular mRNA, snRNA, snoRNA, tRNA, rRNA, gRNA, shRNA, piRNA, rasiRNA, hnRNA, long non-coding RNA, plasmid DNA, ceDNA, minicircle DNA, antisense oligonucleotides (ASOs), DNA viral vectors, viral RNA vectors, and non-viral vectors.

[0049] In some embodiments, the mass ratio of the active ingredient to the lipid nanoparticles is 1:8~12.

[0050] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0051] Experimental methods in the embodiments of this invention that do not specify specific conditions are generally performed under conventional conditions or as recommended by the raw material or product manufacturer. Reagents whose specific source is not specified are commercially available conventional reagents.

[0052]

[0053] Example 1: Preparation of compound ZGB12 Synthesis of Compound 3: 0.35 g (3.43 mmol) of γ-aminobutyric acid was accurately weighed into a 50 mL round-bottom flask, and 5 mL of isopropanol and 1.43 mL (1.042 g, 10.29 mmol) of triethylamine were added. The mixture was then placed on a magnetic stirrer with a clean magnetic stir bar and stirred continuously. Subsequently, 2.79 mL (2.47 g, 10.29 mmol) of dodecyl acrylate was added dropwise to the system. The reaction mixture was stirred and refluxed at 90 °C for 24 h, and the reaction was monitored by thin-layer chromatography until complete. The reaction mixture was cooled to room temperature, concentrated under reduced pressure to remove the solvent, and dried. The crude product was purified by thin-layer chromatography (eluent: methanol and dichloromethane, volume ratio 1:10) to obtain Compound 3, with a yield of 61.5%.

[0054] Synthesis of compound ZGB12: 0.10 g of intermediate product GB12 was accurately weighed and dissolved in a 50 mL round-bottom flask containing 6 mL of dichloromethane. 0.039 g (0.205 mmol) of EDCI and 0.028 g (0.205 mmol) of HOBT were added to the flask, and the mixture was stirred and activated for 30 min. 6.5 μL (0.0063 g, 0.043 mmol) of tris(2-aminoethyl)amine and 0.12 mL (0.089 g, 0.513 mmol) of DIPEA were added to the activated system sequentially. The entire mixture was stirred and reacted at room temperature for 24 h. The reaction was monitored by TLC until complete. The reaction was quenched with saturated sodium bicarbonate solution. The organic phase was extracted with saturated sodium bicarbonate solution and saturated NaCl solution in sequence. The combined organic phase was then dried with anhydrous Na2SO4, filtered, and the organic solvent was removed by rotary evaporation under reduced pressure. The crude reactant was purified by column chromatography (methanol and dichloromethane volume ratio of 1:10) to obtain the final product ZGB12, with a yield of 33.4%. 1H NMR (400 MHz, Chloroform-d) δ6.97(s, 3H), 4.05(t,J=6.8 Hz, 12H), 3.28(q,J=5.8 Hz, 6H), 2.75(t,J=7.1 Hz, 12H), 2.58(q,J=8.7,7.3 Hz, 6H), 2.46-2.40(m,18H), 2.19(t,J=7.2 Hz, 6H), 1.76(t,J=6.9 Hz, 6H), 1.62(q,J=6.9 Hz, 12H), 1.27(d,J=7.4 Hz, 108H), 0.88(t,J=6.7 Hz, 18H). Example 2: Preparation of compounds ZGB8, ZGB10, and ZGB14 Compounds ZGB8, ZGB10, and ZGB14 were prepared using the method described in Example 1, with the following differences: In the preparation of compound ZGB8, γ-aminobutyric acid was used to prepare intermediate compound 3, the organic ester of which was octyl ester, and the product was ZGB8. Structural characterization: 1H NMR(400 MHz, Chloroform-d) δ6.98(s, 3H), 4.31(t,J=7.5 Hz,12H), 3.54(q,J=6.7 Hz, 6H), 2.89(t,J=5.8 Hz, 12H), 2.63(q,J=7.1 Hz, 6H),2.52-2.57(m, 18H),2.36(t,J=6.2 Hz, 6H), 1.75(t,J=7.8 Hz, 6H), 1.64(q,J=6.6Hz, 12H), 1.38(d,J=6.4 Hz, 84H), 0.88(t,J=6.1 Hz, 18H). In the preparation of compound ZGB10, intermediate compound 3 was prepared using γ-aminobutyric acid, with decyl ester as the organic ester, and the product was ZGB10. Structural characterization was as follows: 1 H NMR(400 MHz, Chloroform-d) δ6.84(s, 3H),4.05(t,J=6.8Hz, 12H), 3.37(q,J=6.2 Hz, 6H),2.77(t,J=6.3 Hz, 12H), 2.65(q,J=7.8, 7.1 Hz,6H), 2.48-2.53(m, 18H), 2.25(t,J=6.7 Hz, 6H),1.66(t,J=7.3 Hz, 6H), 1.57(q,J=6.3 Hz, 12H), 1.35(d,J=6.9 Hz, 84H), 0.94(t,J=5.8 Hz, 18H). In the preparation of compound ZGB14, intermediate compound 3 was prepared using γ-aminobutyric acid. The organic ester was tetradecyl ester, and the product was ZGB14. Structural characterization: 1 H NMR(400 MHz, Chloroform-d) δ6.87(t,J=5.9 Hz, 3H),4.05(t,J=6.8 Hz, 12H), 3.55(q,J=6.4 Hz, 6H), 2.85(t,J=6.6 Hz, 6H), 2.71(t,J=6.9 Hz, 12H), 2.36(dt,J=16.5,6.6 Hz, 18H), 2.16(t,J=7.0 Hz, 6H), 1.72(p,J=6.8Hz, 6H), 1.63(q,J=6.9 Hz, 12H), 1.37–1.23(m, 132H),0.88(t,J=6.6 Hz, 18H). Example 3: Preparation of phagocytogenic lipid nanoparticles (DLNPs): The ionizable lipid compound ZGB12, cholesterol, DSPE-PEG2k, DOPE, and the fatty acid bioactive substance docosahexaenoic acid (DHA) prepared in Example 1 were dissolved in anhydrous ethanol to prepare stock solutions with a concentration of 10 mg / ml. After the above materials were prepared, the four raw material stock solutions were mixed and vortexed to homogenize them according to the molar ratio of ionizable lipid:cholesterol:DSPE-PEG2k:DOPE:DHA = 25:20:0.5:7.5:22.5. EGFP (or Luciferase) mRNA was dissolved in disodium hydrogen phosphate-citrate buffer (pH=4.0) to prepare an aqueous mRNA solution. Using a smart LNPs synthesizer, the flow rate of the aqueous phase and the ethanol phase were set to 3:1 to rapidly mix and obtain a solution containing lipid nanoparticles, wherein the mass ratio of mRNA to ZGB12 was 1:10. The LNPs solution prepared by the LNPs synthesizer was dialyzed with PBS buffer for 12 h to remove ethanol, and finally concentrated by centrifugation through an ultrafiltration tube to obtain lipid nanoparticles encapsulating mRNA.

[0055] Example 4: Characterization of DLNP particle size, potential and encapsulation efficiency The morphology of DLNPs was characterized by transmission electron microscopy. The nanoscale size, polydispersity index (PDI), and zeta potential of DLNPs were detected by a dynamic light scattering laser particle size analyzer (Malvern Zetasizer Nano ZS) in 90° backscattering detection mode.

[0056] The encapsulation efficiency of SDLNP-SE was determined using the Ribogreen quantitative assay kit. Efficiency (EE%). First, the undialyzed SDLNP-SE was diluted with 1×TE buffer. 50 μL of SDLNP dilution buffer was added to rows A and B of a black 96-well plate. Then, 50 μL of 1×TE buffer was added to row A, and 50 μL of 2% Triton X-100 buffer was added to row B to lyse the SDLNPs. The mixture from both rows was thoroughly combined and incubated at 37°C for 10 min. After incubation, 100 μL of RiboGreen working solution was added to all sample wells. After incubation in the dark for 5 min, fluorescence intensity was read using a multi-mode microplate detection system with excitation light set to 480 nm and emission light set to 520 nm. The RNA content in the solution before and after demulsification of the lipid nanoparticles was obtained after data processing. The encapsulation efficiency was calculated using the following formula: Encapsulation efficiency (%) = (RNA content after demulsification - RNA content before demulsification) / RNA content after demulsification The results are as follows Figure 1 , Figure 2As shown, DLNPs are spherical particles with an average particle size of 161.87 ± 2.62 nm and a PDI of less than 0.10. The final measured average encapsulation efficiency of DLNPs was 90.80 ± 1.73%, indicating good encapsulation performance. This demonstrates that the prepared DLNPs can successfully cross the intercellular space, effectively encapsulate mRNA, and possess excellent nucleic acid drug delivery performance. Figure 3 As shown, the Zeta potential is -6.04±0.07 mV, which proves that the DLNP formulation system is stable and does not easily aggregate or settle.

[0057] Example 5: Characterization of DLNP transfection efficiency RAW264.7 cells in the logarithmic growth phase were seeded into 96-well plates containing DMEM medium at a density of 1 × 10⁶ cells per well. 5 Cells were collected and, after cell adhesion, prepared for transfection (37°C, approximately 12 hours). Lipid nanoparticles containing 0.2 μg mRNA were added to each well, with three replicates per group. 24 hours after transfection, the transfection efficiency of each group was assessed using flow cytometry. The results are shown below. Figure 2 As shown, the transfection rate is between 60% and 80%.

[0058] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A tri(2-aminoethyl)amine-based ionizable lipid characterized by, The chemical structure is shown as formula I, wherein, R1, R2 are independently selected from substituted or unsubstituted C8-C14 alkyl, substituted or unsubstituted C8-C14 alkenyl, substituted or unsubstituted C8-C14 alkynyl.

2. The ionizable lipid of claim 1, wherein R1, R2 are independently selected from C8-C14 straight chain alkyl.

3. The ionizable lipid of claim 1, wherein selected from the following compounds: 。 4. A process for the preparation of a tri(2-aminoethyl)amine-based ionizable lipid, characterized in that, The steps include the following reaction process: Michael addition reaction of acrylate derivative and gamma-aminobutyric acid to obtain an intermediate; amide reaction of the intermediate and tris(2-aminoethyl)amine to obtain the compound shown as formula I; The chemical structural formula of the acrylate derivative is ; The chemical structural formula of the intermediate is ; R is R1 and / or R2, R1 and / or R2 are independently selected from substituted or unsubstituted C8-C14 alkyl, substituted or unsubstituted C8-C14 alkenyl, substituted or unsubstituted C8-C14 alkynyl.

5. The production method according to claim 4, wherein R1, R2 are independently selected from C8-C14 straight chain alkyl; or, R1, R2 are C8 straight chain alkyl, C10 straight chain alkyl, C12 straight chain alkyl or C14 straight chain alkyl; or, the temperature of the Michael addition reaction is 85~95 ℃; or, the molar ratio of acrylate derivative and gamma-aminobutyric acid is 1:(2.5~3.5); or, EDCI and HOBT are used to activate the carboxyl group of the intermediate in the amide reaction; or, the molar ratio of the intermediate and tris(2-aminoethyl)amine is (3.5~4.5):

1.

6. A lipid nanoparticle characterized by, The ionizable lipid based on tris(2-aminoethyl)amine, the auxiliary lipid, the solid sterol and the PEG lipid according to any one of claims 1~3.

7. The lipid nanoparticle of claim 6, wherein the lipid nanoparticle comprises, Further comprising a fatty acid active substance; preferably, the fatty acid active substance is docosahexaenoic acid; preferably, the mass ratio of the ionizable lipid to the fatty acid active substance is (15~45):(2.5~30); or, the solid sterol is cholesterol; preferably, the molar ratio of the ionizable lipid to the solid sterol is (15~45):(20~35); or, the auxiliary phospholipid is DOPE; preferably, the molar ratio of the ionizable lipid to the auxiliary phospholipid is (15~45):(10~40); or, the PEG lipid is DSPE-PEG2000; preferably, the molar ratio of the ionizable lipid to the PEG lipid is (15~45):(0.5~5).

8. Use of the lipid nanoparticle according to claim 6 or 7 in the preparation of a drug delivery carrier.

9. A pharmaceutical composition, characterized by, It comprises an active ingredient and the lipid nanoparticle according to claim 6 or 7.

10. The pharmaceutical composition of claim 9, wherein The active ingredient is a drug for treating neurodegenerative diseases, cancer, autoimmune diseases, inflammation or genetic diseases; or, the active ingredient is a nucleic acid drug; preferably, the nucleic acid drug comprises one or a combination of siRNA, mRNA, microRNA, circular mRNA, snRNA, snoRNA, tRNA, rRNA, gRNA, shRNA, piRNA, rasiRNA, hnRNA, long non-coding RNA, plasmid DNA, ceDNA, minicircle DNA, antisense oligonucleotide, DNA viral vector, viral RNA vector, non-viral vector. or the mass ratio of the active ingredient and the lipid nanoparticle is 1:8~12.

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