Lipid nanoparticle based on taurochenodeoxycholic acid modification and preparation method and application thereof

Abstract

The invention provides a lipid nanoparticle based on taurochenodeoxycholic acid modification as well as a preparation method and application thereof. The lipid nanoparticle comprises nucleic acid and a lipid carrier encapsulating the nucleic acid, the lipid carrier is prepared from the following components in percentage by mole: 40 to 65 percent of ionized lipid, 5 to 20 percent of structural phospholipid, 20 to 50 percent of sterol component and 0.5 to 5 percent of PEG (Polyethylene Glycol)-lipid, wherein the sterol component comprises cholesterol and taurochenodeoxycholic acid. By replacing cholesterol with taurochenodeoxycholic acid, the membrane order degree and fluidity of the lipid carrier are effectively adjusted, and the targeted delivery and endosome release efficiency of LNP to liver cells is remarkably improved, so that an LNP delivery system can obtain a more remarkable and lasting Lp (a) reduction effect under the same or lower dosage; by optimizing the LNP formula, it is ensured that the LNP preparation has higher controllability and reliability in the production and storage process.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a lipid nanoparticle modified with taurine chenodeoxycholic acid, its preparation method, and its application. Background Technology

[0002] Apolipoprotein(a) [Lp(a)] is an independent risk factor for atherosclerotic cardiovascular disease. Its level is mainly determined by LPA gene polymorphism. Existing lipid-lowering drugs, such as statins, lower cholesterol by inhibiting HMG-CoA reductase, but are almost ineffective against Lp(a).

[0003] RNA interference technology offers a novel strategy for targeting the LPA gene. Small interfering RNA (siRNA) can silence LPA gene expression at the posttranscriptional level. However, the core bottleneck in successfully developing LPA siRNA into drugs has shifted from sequence discovery to delivery systems.

[0004] Lipid nanoparticles (LNPs) are currently the most clinically validated and mature nucleic acid delivery platform. A typical LNP consists of ionizable lipids, helper phospholipids, sterols, and PEGylated lipids, with cholesterol being an essential sterol component for maintaining LNP stability. Traditional LNPs using cholesterol as the sole sterol suffer from insufficient hepatic uptake efficiency, high dose burden, and limited stability and batch-to-batch consistency when delivering LPA siRNA. Therefore, developing a lipid nanoparticle system and its preparation method that can efficiently, stably, and scalably deliver siRNA to the target gene Lp(a) into hepatocytes without significantly increasing process complexity and toxicity is a pressing technical problem in this field. Summary of the Invention

[0005] In view of the aforementioned problems, this application is made to provide a lipid nanoparticle based on taurine chenodeoxycholic acid modification, a method for its preparation, and its application, which overcomes or at least partially solves the aforementioned problems. A lipid nanoparticle based on taurine chenodeoxycholic acid modification, comprising nucleic acid and a lipid carrier encapsulating the nucleic acid; The lipid carrier comprises, by molar percentage: 40%-65% ionized lipids, 5%-20% structured phospholipids, 20%-50% sterol components and 0.5%-5% PEG-esters; wherein the sterol components include cholesterol and taurine chenodeoxycholic acid.

[0006] Preferably, the taurine chenodeoxycholic acid accounts for 0.1%-100% of the total molar amount of the sterol components.

[0007] Preferably, the nitrogen-phosphorus molar ratio of the ionized lipid to the nucleic acid is 3:1 to 10:1.

[0008] Preferably, the nucleic acid is siRNA.

[0009] Preferably, the ionized lipids include one or more of DLin-MC3-DMA, 5A2-SC8, SM-102, C12-200, ALC-0315, DODAP, and DODMA.

[0010] Preferably, the structural phospholipids include neutral accessory phospholipids; the neutral accessory phospholipids include, but are not limited to, 1,2-distearyl-sn-glycerol-3-phosphate choline, 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline, 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-palmitoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dimyristoyl-sn-glycerol-3-phosphate ethanolamine, 2-dioleoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol), oleoylphosphatidylcholine, and 1-palmitoyl-2-oleoylphosphatidylethanolamine.

[0011] Preferably, the PEG-ester includes one or more of ALC-0159, DSPE-PEG2000, and DMG-PEG2000.

[0012] This application also provides a method for preparing lipid nanoparticles modified with taurine chenodeoxycholic acid, comprising the following steps: The ionizable lipid, the auxiliary phospholipid, the sterol component, and the polyethylene glycol-modified lipid are dissolved in an organic solvent according to a first preset ratio to obtain a lipid organic phase; The nucleic acid is dissolved in a buffer solution according to the second preset ratio to obtain an aqueous phase of nucleic acid; The lipid organic phase is mixed with the nucleic acid aqueous phase to allow the lipids to self-assemble into a crude dispersion of lipid nanoparticles encapsulated with the nucleic acid. The crude dispersion of the lipid nanoparticles was dialyzed to replace the solvent and neutralize the pH, thereby obtaining the target lipid nanoparticles.

[0013] This application also provides the use of the above-mentioned lipid nanoparticles in the preparation of a drug for reducing serum lipoprotein(a) [Lp(a)] levels; The drug is indicated for the prevention or treatment of diseases associated with elevated Lp(a), which are selected from atherosclerotic cardiovascular diseases, ischemic heart disease, ischemic stroke, peripheral artery disease and / or calcific aortic stenosis. Preferably, the administration methods of the drug include, but are not limited to, oral, enteral, subcutaneous, intramuscular, intravenous, nasal, transdermal, subconjunctival, intraocular, orbital, retroocular, retinal, choroidal, and intrathecal administration. Preferably, the dosage form of the drug includes, but is not limited to, tablets, capsules, pills, injections, inhalers, lozenges, suppositories, emulsions, microemulsions, submicroemulsions, nanoparticles, gels, powders, suspensions, creams, gels, and sprays.

[0014] This application also provides the use of the above-described lipid nanoparticles, the lipid nanoparticles prepared by the above-described preparation method, and the above-described drug in any of the following: 1) Application in the preparation of drugs for the treatment and / or prevention of diseases related to LPA gene expression; 2) Application in suppressing LPA gene expression.

[0015] Preferably, the method of using the drug is as follows: by using lipid nanoparticles as described above, lipid nanoparticles prepared by the preparation method described above, or the drug as described above to inhibit the LPA target activity or reduce the LPA expression level in the subject. Preferably, the diseases associated with LPA gene expression include atherosclerotic cardiovascular disease, ischemic heart disease, ischemic stroke, peripheral artery disease, and / or calcific aortic stenosis.

[0016] This application has the following advantages: In the embodiments of this application, addressing the problems of insufficient liver uptake efficiency, high dose burden, and limited stability and batch-to-batch consistency in the prior art, this application provides a solution based on taurine chenodeoxycholic acid modified lipid nanoparticles, specifically comprising: nucleic acid and a lipid carrier encapsulating the nucleic acid; the lipid carrier, by molar percentage, comprises: 40%-65% ionized lipids, 5%-20% structured phospholipids, 20%-50% sterol components and 0.5%-5% PEG-esters; wherein the sterol components include cholesterol and taurine chenodeoxycholic acid. By replacing cholesterol with TCDCA (taurine chenodeoxycholic acid), the membrane order and fluidity of LNP (liposome carrier) were effectively regulated, significantly improving the targeted delivery and endosomal release efficiency of LNP to hepatocytes. This enabled the LNP delivery system to achieve a more significant and sustained Lp(a) reduction effect at the same or lower doses. Optimizing the LNP formulation made the particle size, PDI, and encapsulation efficiency of LNP more stable, improving batch-to-batch consistency and ensuring higher controllability and reliability of LNP formulations during production and storage. Attached Figure Description

[0017] To more clearly illustrate the technical solution of this application, the drawings used in the description of this application will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 This is a schematic flowchart of a method for preparing lipid nanoparticles modified with taurine chenodeoxycholic acid according to an embodiment of this application. Figure 2 This is a flowchart illustrating a method for preparing lipid nanoparticles modified with taurine chenodeoxycholic acid, as provided in one embodiment of this application. Figure 3 This is a real-time fluorescence quantitative PCR result diagram provided in an embodiment of the present invention; Figure 4 This is a protein immunoblotting result image provided in an embodiment of the present invention; Figure 5 This is a mouse in vivo fluorescence imaging image provided in an embodiment of the present invention; Figure 6 This is an ELISA graph showing the change in Lp(a) levels in mouse plasma provided in an embodiment of the present invention. Detailed Implementation

[0019] To make the objectives, features, and advantages of this application more apparent and understandable, the application will be further described in detail below with reference to the accompanying drawings and specific embodiments. Obviously, the described embodiments are only some, not all, of the embodiments of this application. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application.

[0020] The inventors discovered through analysis of existing technologies that traditional LNPs, which use cholesterol as the sole sterol, have the following drawbacks when delivering LPA siRNA: (1) Insufficient hepatocyte delivery efficiency: Limited cell uptake and endosomal escape capacity, resulting in the need for high doses or frequent administration to maintain efficacy, increasing toxicity risk and treatment cost.

[0021] (2) Poor formulation stability and batch-to-batch consistency: Cholesterol-dependent LNPs are prone to particle size drift and encapsulation rate fluctuations during self-assembly, making quality control difficult in large-scale production.

[0022] (3) Single structure and function: Cholesterol has limited ability to regulate membrane fluidity and membrane curvature, making it difficult to achieve a balance between circulatory stability and endosome dissociation.

[0023] One embodiment of this application provides a lipid nanoparticle modified with taurine chenodeoxycholic acid, comprising nucleic acid and a lipid carrier encapsulating the nucleic acid; The lipid carrier comprises, by molar percentage: 40%-65% ionized lipids, 5%-20% structured phospholipids, 20%-50% sterol components and 0.5%-5% PEG-esters; wherein the sterol components include cholesterol and taurine chenodeoxycholic acid.

[0024] In the embodiments of this application, addressing the problems of insufficient hepatic uptake efficiency, high dose burden, and limited stability and batch-to-batch consistency in the prior art, this application provides a solution based on taurine chenodeoxycholic acid-modified lipid nanoparticles. By using TCDCA to replace cholesterol, the membrane order and fluidity of LNPs are effectively regulated, significantly improving the targeted delivery and endosomal release efficiency of LNPs to hepatocytes. This allows the LNP delivery system to achieve a more significant and sustained Lp(a) reduction effect at the same or lower doses. By optimizing the LNP formulation, the particle size, PDI, and encapsulation efficiency of LNPs are made more stable, improving batch-to-batch consistency and ensuring higher controllability and reliability of LNP formulations during production and storage.

[0025] The following will further describe a lipid nanoparticle modified with taurine chenodeoxycholic acid in this exemplary embodiment.

[0026] In one embodiment of the present invention, the taurine chenodeoxycholic acid accounts for 0.1%-100% of the total molar amount of the sterol component.

[0027] It should be noted that TCDCA (taurine chenodeoxycholic acid) is an amphiphilic bile acid compound formed by the combination of chenodeoxycholic acid and taurine. Its molecular structure combines a hydrophobic steroidal core skeleton with a hydrophilic taurine side chain. This amphiphilic nature allows it to form interfacial interactions between the aqueous and lipid phases, while also exhibiting good biocompatibility. It can be metabolized by the liver in vivo and has low toxicity. TCDCA can regulate LNP membrane behavior, reduce membrane defects, and improve encapsulation and storage stability; it can also synergistically promote endosome membrane rupture with ionized lipids, enhancing the efficiency of siRNA cytoplasmic release.

[0028] As an example, the sterol component consists of cholesterol and TCDCA, with the TCDCA substitution ratio being 0.1%, 5%, 25%, 50%, 75%, or 90%.

[0029] In one specific implementation, taurine chenodeoxycholic acid accounts for 25% of the total molar amount of the sterol component. This proportion allows the LNP carrier membrane to be in the tuning region between the cholesterol-dominated and bile acid-dominated phases, maintaining the orderly packaging and permeation barrier of the laminar phase while introducing appropriate membrane fluidity. This achieves improved efficacy without increasing process complexity or the quality of ionized lipids, while also having good tolerance for process parameters, supporting flexible adjustment of clinical dosage and dosing frequency, and facilitating individualized treatment.

[0030] In one embodiment of the present invention, the nitrogen-phosphorus molar ratio of the ionized lipid to the nucleic acid is 3:1 to 10:1.

[0031] It should be noted that ionized lipids are a type of pH-responsive cationic lipids. Their molecular structure contains protonable amino groups and hydrophobic fatty acid segments, which have good lipid solubility and can participate in the self-assembly of LNPs. Due to their pH responsiveness, they can become carriers for siRNA delivery.

[0032] The nitrogen / phosphorus (N / P) molar ratio is the electrostatic interaction between the nitrogen atoms of ionized lipids and the phosphorus atoms of nucleic acids, which is the core force for lipid carriers to encapsulate nucleic acids.

[0033] As an example, the stoichiometry between ionized lipids and oligonucleotides (such as siRNA) is set at N / P = 3-10, preferably 6.

[0034] In one embodiment of the present invention, the nucleic acid is siRNA. Specifically, small interfering RNA (siRNA) technology is currently used to inhibit the expression of specific genes. It is a synthetically produced double-stranded RNA fragment of about 20 base pairs. After entering the cell, the double strands dissociate, and one strand specifically binds to the messenger RNA (mRNA) of the target gene through complementary base pairing. The mRNA is then degraded by the widely distributed RNA-induced silencing complex (RISC) mechanism in cells, thereby inhibiting the expression of the target gene. Through modification and delivery systems, siRNA can enter human cells as a drug and specifically inhibit the expression of some pathogenic genes.

[0035] In one embodiment of the present invention, the ionized lipids include one or more of DLin-MC3-DMA, 5A2-SC8, SM-102, C12-200, ALC-0315, DODAP, and DODMA.

[0036] In one specific implementation, the ionized lipid is SM-102, which has good liver targeting properties and low immunogenicity. It is added to the lipid carrier at a molar percentage of 50% to synergistically achieve efficient encapsulation and delivery of siRNA with other components.

[0037] In one embodiment of the present invention, the structural phospholipid includes neutral accessory phospholipids; the neutral accessory phospholipids include, but are not limited to, 1,2-distearyl-sn-glycerol-3-phosphate choline, 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline, 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-palmitoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dimyristoyl-sn-glycerol-3-phosphate ethanolamine, 2-dioleoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol), oleoylphosphatidylcholine, and 1-palmitoyl-2-oleoylphosphatidylethanolamine.

[0038] Specifically, structural phospholipids are natural or synthetic phospholipid compounds that are fundamental components of biological membranes and artificial lipid membranes. Their molecules possess both a hydrophilic phosphate head and a hydrophobic fatty acid chain, making them amphiphilic molecules with excellent biocompatibility. The structural phospholipids (or accessory phospholipids) described in this embodiment are selected from one or more of natural phospholipids, synthetic phospholipids, and their derivatives. Specifically, they may include phosphatidylcholine (PC), phosphatidylethanolamine (PE), or combinations thereof; preferably one or more of distearylphosphatidylcholine (DSPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylethanolamine (DOPE), soybean lecithin, egg yolk lecithin, and hydrogenated soybean lecithin (HSPC).

[0039] In one embodiment of the present invention, the PEG-lipid includes one or more of ALC-0159, DSPE-PEG2000, and DMG-PEG2000. Specifically, the PEG-lipid is a polyethylene glycol-modified lipid grafted onto the surface of lipid nanoparticles, which can form a steric hindrance layer, reduce the immune clearance of nanoparticles, prolong the in vivo circulation time, and simultaneously regulate the particle size and dispersibility of lipid nanoparticles.

[0040] Reference Figure 1 This illustration shows a method for preparing lipid nanoparticles modified with taurine chenodeoxycholic acid according to an embodiment of this application. Including the following steps: S110. Dissolve the ionizable lipid, the auxiliary phospholipid, the sterol component and the polyethylene glycol-modified lipid in an organic solvent according to the first preset ratio to obtain a lipid organic phase; S120. Dissolve the nucleic acid in a buffer solution according to the second preset ratio to obtain an aqueous phase of nucleic acid; S130. The lipid organic phase is mixed with the nucleic acid aqueous phase to allow the lipids to self-assemble into a crude dispersion of lipid nanoparticles encapsulated with the nucleic acid. S140. Dialyze the crude dispersion of the lipid nanoparticles to replace the solvent and neutralize the pH, thereby obtaining the target lipid nanoparticles.

[0041] In one embodiment of the present invention, the specific process of step S110, "dissolving the ionizable lipid, the auxiliary phospholipid, the sterol component and the polyethylene glycol-modified lipid in an organic solvent according to a first preset ratio to obtain a lipid organic phase" can be further described in conjunction with the following description.

[0042] According to the molar percentage, 40%-65% of ionized lipids, 5%-20% of structural phospholipids, 20%-50% of sterol components, and 0.5%-5% of PEG-esters are dissolved in an organic solvent. The organic solvent can be anhydrous ethanol, methanol, or isopropanol. The total lipid concentration is preferably 12-20 mM. The degree of dissolution is such that each lipid component is uniformly dispersed in the organic solvent.

[0043] In one embodiment of the present invention, the specific process of "dissolving the nucleic acid in a buffer solution according to a second preset ratio to obtain a nucleic acid aqueous phase" in step S120 can be further described in conjunction with the following description.

[0044] Oligonucleotides were dissolved in 10-50 mM citrate or acetate buffer to form an aqueous phase. The pH of the buffer was 3.5-5.5, preferably pH 4.0. The stoichiometric relationship between oligonucleotides and ionized lipids was determined based on N / P = 3-10.

[0045] In one embodiment of the present invention, the specific process of step S130, "mixing the lipid organic phase with the nucleic acid aqueous phase to allow the lipids to self-assemble into a crude dispersion of lipid nanoparticles encapsulating the nucleic acid," can be further explained in conjunction with the following description.

[0046] At room temperature and with magnetic stirring at 300-800 rpm, the organic and aqueous phases are mixed to reduce the ethanol content in the system to approximately 30-60% (v / v), resulting in immediate self-assembly and the formation of a crude LNP dispersion. This homogeneous mixture of the organic and aqueous phases triggers the lipid self-assembly effect. Due to their amphiphilic nature, the lipids spontaneously form a core-shell structure in the aqueous environment, encapsulating negatively charged nucleic acids within them through electrostatic interactions, thus forming a crude dispersion. The mixing process requires control of the stirring rate and the organic solvent content in the system.

[0047] In one embodiment of the present invention, the specific process of step S140, "dialysis of the crude dispersion of the lipid nanoparticles to replace the solvent and neutralize the pH to obtain the target lipid nanoparticles," can be further described in conjunction with the following description.

[0048] The crude dispersion of lipid nanoparticles was dialyzed with dialysis fluid for 4-8 hours to reduce the volume fraction of organic residue in the system to less than 10%.

[0049] Specifically, the obtained dispersion is placed into a pre-washed dialysis bag (molecular weight cutoff MWCO 10-14 kDa, or an equivalent specification of 3.5-100 kDa). A buffer solution of pH 7.2-7.5 (e.g., 1xPBS or 10 mM HEPES) is used as the external dialysate. Dialysis is performed at room temperature (20-25°C) for approximately 6 hours, changing the external dialysate 2-4 times during this period to complete solvent replacement and neutralization, ensuring the ethanol volume fraction of the system is <10% (preferably <2%). If necessary, an isotonic adjuster (such as 5% glucose or isotonic NaCl) is added, followed by sterile filtration through a 0.22 μm low-protein adsorption membrane and volume adjustment to obtain the target formulation.

[0050] The semi-permeable nature of the dialysis membrane enables the removal of organic solvents and the neutralization of the system's pH, while retaining lipid nanoparticles. Dialysis can improve the stability and biocompatibility of lipid nanoparticles, laying the foundation for subsequent productization.

[0051] The lipid nanoparticles prepared by the method of this invention have a particle size of 100-200 nm, PDI≤0.20, encapsulation efficiency>70%, and controllable ethanol residue; the above indicators can be repeatedly obtained without changing the existing equipment and operating window.

[0052] This application also provides the use of lipid nanoparticles in pharmaceuticals for reducing serum lipoprotein(a) [Lp(a)] levels; The drug is indicated for the prevention or treatment of diseases associated with elevated Lp(a), which are selected from atherosclerotic cardiovascular diseases, ischemic heart disease, ischemic stroke, peripheral artery disease and / or calcific aortic stenosis. Preferably, the administration methods of the drug include, but are not limited to, oral, enteral, subcutaneous, intramuscular, intravenous, nasal, transdermal, subconjunctival, intraocular, orbital, retroocular, retinal, choroidal, and intrathecal administration. Preferably, the dosage form of the drug includes, but is not limited to, tablets, capsules, pills, injections, inhalers, lozenges, suppositories, emulsions, microemulsions, submicroemulsions, nanoparticles, gels, powders, suspensions, creams, gels, and sprays.

[0053] This application also provides the use of the above-described lipid nanoparticles, the lipid nanoparticles prepared by the above-described preparation method, and the above-described drug in any of the following: 1) Application in the preparation of drugs for the treatment and / or prevention of diseases related to LPA gene expression; 2) Application in suppressing LPA gene expression.

[0054] Preferably, the method of using the drug is as follows: by using lipid nanoparticles as described above, lipid nanoparticles prepared by the preparation method described above, or the drug as described above to inhibit the LPA target activity or reduce the LPA expression level in the subject. Preferably, the diseases associated with LPA gene expression include atherosclerotic cardiovascular disease, ischemic heart disease, ischemic stroke, peripheral artery disease, and / or calcific aortic stenosis.

[0055] Reference Figure 2 This application provides a design flowchart for a method of preparing lipid nanoparticles modified with taurine chenodeoxycholic acid. The main line of this application is "siRNA → formulation → raw materials → self-assembly / replacement → finished product → quality control → pharmacodynamic distribution → dosage optimization": Steps 1 and 2 respectively complete the design of the nucleic acid and vector ends, which are then combined in step 3; LNPs are formed and neutralized in step 4; one product from step 4 enters step 5 for finished product preparation, and the other enters step 6 for quality control; those meeting the release criteria proceed to step 7 for pharmacodynamic and distribution evaluation and step 8 for dosage / dosing frequency optimization, forming a closed loop.

[0056] The following are specific examples: (1) siRNA prediction The full-length sequence and cDNA sequence of the human LPA gene were obtained from the NCBI gene database. Based on a method incorporating machine learning algorithms and a whole-genome siRNA biological reagent library, siRNA sequences that may inhibit LPA expression were predicted. 300 sequences meeting the matching and stability requirements were obtained, and the top 20 sequences were selected for experimental validation based on their scores. The selected sequences are shown in Table 1.

[0057] Table 1 (2) siRNA screening 1) Determine the screening model To assess the siRNA inhibition efficiency, cell lines highly expressing lpa were selected as screening models. Cell line database searches identified RT4 and SNU1066 as candidates, and their expression capabilities were experimentally verified.

[0058] The experimental design is shown in Table 2:

[0059] Table 2 The data and results analysis are shown in Tables 3 and 4:

[0060] Table 3

[0061] Table 4 GAPDH was used as an internal control. A higher CT value indicates a weaker corresponding expression ability. The results showed that the two cell types can be used as cell models for siRNA screening, with RT4 being superior.

[0062] 2) Determine the conditions for transformation Transfection reagents are required for siRNA to enter cells, and the amount of siRNA and transfection reagent used needs to be determined. Therefore, a known positive LPA-siRNA sequence was used as the PC to explore the conditions for chemical transformation.

[0063] The experimental design is shown in Table 5:

[0064] Table 5 The data and results analysis are shown in Table 6:

[0065] Table 6 The results showed that transfection could be completed after 48 hours of culture with RNAiMAX. The positive drug showed LPA expression inhibition at both 3 nM and 30 nM concentrations. Subsequently, siRNA screening was performed at 30 nM.

[0066] 3) siRNA screening The knockout efficiency of the 20 predicted siRNAs was validated using the RT4 cell line. The knockout efficiency is shown in Table 7.

[0067] Table 7 Five sequences (siRNA3, siRNA2, siRNA20, siRNA4, and siRNA17) with a knockout efficiency greater than 40% were selected as potential drug components for further validation.

[0068] (3) Safety and efficacy assessment of siRNA The knockout efficiency was repeatedly verified in the RT4 cell line.

[0069] The experimental design is shown in Table 8:

[0070] Table 8 The data and results analysis are shown in Table 9:

[0071] Table 9 Verification of knockout efficiency in primary monkey hepatocytes: Considering that LPA is mainly expressed by hepatocytes in humans, and that cynomolgus monkeys are closely related to humans, primary monkey hepatocytes were used to verify the knockout efficiency. 1) Probe design The LPA gene differs between monkeys and humans, necessitating the design of primers and probes for qPCR validation of knockout efficiency.

[0072] The data and results analysis are shown in Table 10:

[0073] Table 10 The results showed that the designed probe could be used for the detection of LPA expression in monkeys.

[0074] 2) Knockout efficiency verification The data and results analysis are shown in Table 11:

[0075] Table 11 The results showed that siRNA3, siRNA2 and siRNA20 at 30 nM and 10 nM exhibited consistent and good LPA expression inhibition in both cell lines, and these three sequences were subsequently selected as drug prototypes.

[0076] (4) Validate knockout efficiency and drug distribution in mouse models that overexpress humanized LPA.

[0077] The knockout efficiency was verified in a mouse model overexpressing humanized LPA. Figures 3-5 As shown: Figure 3 This is a graph showing the results of real-time quantitative PCR. Figure 4 This is a graph of the results of Western blot analysis of proteins. Figure 5 This is a live fluorescence imaging image of a mouse.

[0078] (5) Changes in mouse plasma Lp(a) levels (ELISA) Figure 6 As shown.

[0079] Although preferred embodiments of the present application have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of the embodiments of the present application.

[0080] Finally, it should be noted that in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or terminal device that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or terminal device. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or terminal device that includes said element.

[0081] The above provides a detailed description of the lipid nanoparticles modified with taurine chenodeoxycholic acid, their preparation method, and applications. Specific examples have been used to illustrate the principles and implementation methods of this application. The descriptions of the above embodiments are only for the purpose of helping to understand the method and core ideas of this application. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the ideas of this application. Therefore, the content of this specification should not be construed as a limitation of this application.

Claims

1. A lipid nanoparticle modified with taurine chenodeoxycholic acid, characterized in that, Includes nucleic acids and lipid carriers that encapsulate the nucleic acids; The lipid carrier comprises, by molar percentage: 40%-65% ionized lipids, 5%-20% structured phospholipids, 20%-50% sterol components and 0.5%-5% PEG-esters; wherein the sterol components include cholesterol and taurine chenodeoxycholic acid.

2. The lipid nanoparticles according to claim 1, characterized in that, The taurine chenodeoxycholic acid accounts for 0.1%-100% of the total molar amount of the sterol components.

3. The lipid nanoparticles according to claim 1, characterized in that, The nitrogen-phosphorus molar ratio of the ionized lipid to the nucleic acid is 3:1 to 10:

1.

4. The lipid nanoparticles according to claim 1, characterized in that, The nucleic acid in question is siRNA.

5. The lipid nanoparticles according to claim 1, characterized in that, The ionized lipids include one or more of DLin-MC3-DMA, 5A2-SC8, SM-102, C12-200, ALC-0315, DODAP, and DODMA.

6. The lipid nanoparticles according to claim 1, characterized in that, The structural phospholipids include neutral accessory phospholipids; the neutral accessory phospholipids include, but are not limited to, 1,2-distearyl-sn-glycerol-3-phosphate choline, 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline, 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-palmitoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dimyristoyl-sn-glycerol-3-phosphate ethanolamine, 2-dioleoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol), oleoylphosphatidylcholine, and 1-palmitoyl-2-oleoylphosphatidylethanolamine.

7. The lipid nanoparticles according to claim 1, characterized in that, The PEG-ester includes one or more of ALC-0159, DSPE-PEG2000, and DMG-PEG2000.

8. A method for preparing lipid nanoparticles modified with taurine chenodeoxycholic acid, characterized in that, Including the following steps: The ionizable lipid, the auxiliary phospholipid, the sterol component, and the polyethylene glycol-modified lipid are dissolved in an organic solvent according to a first preset ratio to obtain a lipid organic phase; The nucleic acid is dissolved in a buffer solution according to the second preset ratio to obtain an aqueous phase of nucleic acid; The lipid organic phase is mixed with the nucleic acid aqueous phase to allow the lipids to self-assemble into a crude dispersion of lipid nanoparticles encapsulated with the nucleic acid. The crude dispersion of the lipid nanoparticles was dialyzed to replace the solvent and neutralize the pH, thereby obtaining the target lipid nanoparticles.

9. Use of the lipid nanoparticles according to any one of claims 1-7 in the preparation of a medicament for reducing serum lipoprotein(a) [Lp(a)] levels; The drug is indicated for the prevention or treatment of diseases associated with elevated Lp(a), which are selected from atherosclerotic cardiovascular diseases, ischemic heart disease, ischemic stroke, peripheral artery disease and / or calcific aortic stenosis. Preferably, the administration methods of the drug include, but are not limited to, oral, enteral, subcutaneous, intramuscular, intravenous, nasal, transdermal, subconjunctival, intraocular, orbital, retroocular, retinal, choroidal, and intrathecal administration. Preferably, the dosage form of the drug includes, but is not limited to, tablets, capsules, pills, injections, inhalers, lozenges, suppositories, emulsions, microemulsions, submicroemulsions, nanoparticles, gels, powders, suspensions, creams, gels, and sprays.

10. The use of the lipid nanoparticles according to any one of claims 1-7, the lipid nanoparticles prepared by the preparation method according to claim 8, and the drug according to claim 9 in any one of the following: 1) Application in the preparation of drugs for the treatment and / or prevention of diseases related to LPA gene expression; 2) Application in suppressing LPA gene expression; Preferably, the method of using the drug is as follows: by using the lipid nanoparticles as described in claims 1-7, the lipid nanoparticles prepared by the preparation method as described in claim 8, or the drug as described in claim 9 to inhibit the LPA target activity or reduce the LPA expression level in the subject; Preferably, the diseases associated with LPA gene expression include atherosclerotic cardiovascular disease, ischemic heart disease, ischemic stroke, peripheral artery disease, and / or calcific aortic stenosis.

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