A liver-kidney-targeted nucleic acid-lipid nanoparticle, a preparation method and application thereof

Abstract

This invention relates to a nucleic acid-lipid nanoparticle for enhanced liver and kidney targeting, its preparation method, and its application. The nucleic acid-lipid nanoparticle comprises lipid nanoparticles and a nucleic acid drug encapsulated therein. The raw materials for preparing the lipid nanoparticles include myristoylphosphatidylcholine, ionizable lipids, cholesterol, and triglycerides. The nucleic acid drug is an siRNA drug. Based on the crucial role of apolipoprotein E in liver-specific recognition and endocytosis, this invention enhances and predictably regulates liver delivery efficiency by modulating the interaction between the molecular structure of helper phospholipids in the lipid nanoparticles and apolipoprotein E, providing a general and scalable design strategy for liver-targeted delivery of nucleic acid drugs.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a nucleic acid-lipid nanoparticle that enhances liver and kidney targeting, its preparation method, and its application. Background Technology

[0002] Lipid nanoparticles (LNPs) are a class of nanocarriers commonly used for nucleic acid drug delivery. They can encapsulate nucleic acid molecules to protect them from in vivo degradation and promote their uptake and release into the cytoplasm by cells. Existing LNP systems typically consist of components such as ionizable lipids, auxiliary lipids, cholesterol, and polyethylene glycol (PEG) lipids, and their composition and proportions can be adjusted according to delivery requirements.

[0003] Existing research indicates that liver-derived plasma nutrients (LNPs) often exhibit organ-biased distribution after in vivo administration, particularly accumulating in the liver. This process is closely related to the physicochemical properties of LNPs and the biomolecular crown formed by their interaction with plasma proteins in vivo, with some plasma proteins (such as apolipoproteins) believed to be involved in the hepatic uptake of LNPs. Based on these characteristics, existing technologies have attempted to improve the hepatic delivery capacity of LNPs by modulating their composition or surface properties.

[0004] However, existing LNP liver delivery strategies still mainly rely on empirical formulation optimization or non-specific in vivo distribution characteristics, making it difficult to achieve stable and predictable liver delivery results under different nucleic acid types or application scenarios. Furthermore, current technologies lack effective and scalable solutions for improving liver delivery efficiency while maintaining the universality of the LNP system.

[0005] Therefore, it is necessary to provide a new lipid nanoparticle technology solution to overcome the shortcomings of existing technologies in terms of liver delivery efficiency and applicability. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a nucleic acid-lipid nanoparticle that enhances liver and kidney targeting, its preparation method, and its application.

[0007] To achieve this objective, the present invention adopts the following technical solution: In a first aspect, the present invention provides a nucleic acid-lipid nanoparticle that enhances liver and kidney targeting, wherein the nucleic acid-lipid nanoparticle comprises lipid nanoparticles and a nucleic acid drug encapsulated therein; the raw materials for preparing the lipid nanoparticles include myristoylphosphatidylcholine, ionizable lipids, cholesterol and triglycerides; and the nucleic acid drug is an siRNA drug.

[0008] This invention creatively designs a lipid nanoparticle delivery system in which dimyristoylphosphatidylcholine (DMPC), ionizable lipids, cholesterol, and triglycerides synergistically work together. This allows the lipid nanoparticles of this invention to efficiently load nucleic acid drugs and actively target the liver or kidneys for efficient delivery, even without polyethylene glycol modification. Based on the crucial role of apolipoprotein E (ApoE) in liver-specific recognition and endocytosis, this invention enhances and predictably regulates liver delivery efficiency by modulating the interaction between the molecular structure of helper phospholipids in the lipid nanoparticles and ApoE. This provides a general and scalable design strategy for liver-targeted delivery of nucleic acid drugs.

[0009] Preferably, the molar percentages of each component in the raw materials for preparing the lipid nanoparticles are: 5%-10% myristoyl phosphatidylcholine (e.g., 5%, 6%, 7%, 8%, 9%, 10%, etc.), 15%-25% ionizable lipids (e.g., 15%, 17%, 19%, 21%, 23%, 25%, etc.), 15%-25% cholesterol (e.g., 15%, 17%, 19%, 21%, 23%, 25%, etc.), and 45%-60% triglycerides (e.g., 45%, 47%, 50%, 53%, 55%, 57%, 60%, etc.).

[0010] Preferably, the ionizable lipids include any one or a combination of at least two of DLin-MC3-DMA, DLin-DMA, and DLin-KC2-DMA.

[0011] Preferably, the ionizable lipid is DLin-MC3-DMA.

[0012] Preferably, the triglyceride includes any one or a combination of at least two of tricaprylic acid glyceride, tricaprylic acid glyceride, and triheptanoic acid glyceride.

[0013] Preferably, the triglyceride is trioctanoic acid glyceride.

[0014] This invention creatively replaces PEGylated lipids in traditional LNP formulations with medium-chain triglycerides (such as tricaprylyl glycerol). On the one hand, triglycerides, as an oily core, help improve the encapsulation efficiency and stability of nucleic acid drugs; on the other hand, it avoids the "PEG dilemma" (accelerated blood clearance phenomenon) that may be caused by PEGylated lipids, and unexpectedly found that the synergistic effect of triglycerides and DMPC can more effectively enrich apolipoprotein E, thereby significantly enhancing liver targeting.

[0015] Preferably, the siRNA drug is a sequence that targets the complement component C3 gene.

[0016] Preferably, the sequence of the siRNA drug is selected from any one or a combination of at least two of the following sequences: (1) The amino acid sequence shown in SEQ ID No. 1-SEQ ID No. 3, or, An amino acid sequence obtained by substituting, deleting, or adding 1-3 amino acid residues to the sequence described in (1), and having the same or similar function to the sequence described in (1), or, (3) An amino acid sequence that has at least 95% sequence homology with the sequence described in (1) or (2) and has the same or similar function as the sequence described in (1).

[0017] Preferably, the siRNA drug is a mixture of sequences SEQ ID No. 1-SEQ ID No. 3.

[0018] The gene sequences of SEQ ID No. 1-SEQ ID No. 3 are shown below: SEQ ID No.1: CCAAGAATCGCTACTTCCA; SEQ ID No.2: CCCTCATCATCTACCTAGA; SEQ ID No. 3: CCGAGCTAACCAACTAGA.

[0019] This invention creatively uses a mixture of three sequences as a nucleic acid drug, which can achieve better gene knockout effects than using any single sequence of the nucleic acid drug alone.

[0020] Preferably, the nitrogen-to-phosphorus ratio of the ionizable lipid and siRNA is (4-8):1 (e.g., it can be 4:1, 5:1, 6:1, 7:1, 8:1, etc.).

[0021] Preferably, the nucleic acid-lipid nanoparticles have a particle size of 140-155 nm (e.g., 140 nm, 142 nm, 145 nm, 147 nm, 150 nm, 153 nm, 155 nm, etc.).

[0022] In a second aspect, the present invention provides a method for preparing nucleic acid-lipid nanoparticles as described in the first aspect, the method comprising: (1) Mix dimyristoyl phosphatidylcholine, ionizable lipids, cholesterol, triglycerides and organic solvents to obtain a lipid phase; The siRNA was mixed with an acidic buffer to obtain the aqueous phase. (2) Using a microfluidic device, the lipid phase and the aqueous phase are mixed to obtain the nucleic acid-lipid nanoparticles.

[0023] Preferably, the mixing temperature in step (1) is 20-30℃ (e.g., 20℃, 22℃, 24℃, 26℃, 28℃, 30℃, etc.).

[0024] Preferably, the organic solvent in step (1) includes any one or a combination of at least two of methanol, ethanol, isopropanol, and n-butanol.

[0025] Preferably, the acidic buffer solution in step (1) includes any one or a combination of at least two of citrate buffer, acetate buffer, and succinate buffer.

[0026] Preferably, the mixing temperature in step (2) is 20-30℃ (e.g., 20℃, 22℃, 24℃, 26℃, 28℃, 30℃, etc.).

[0027] Preferably, the volumetric flow rate of the lipid phase and aqueous phase mixture in step (2) is (2-5):1 (e.g., 2:1, 3:1, 4:1, 5:1, etc.), and the total flow rate is 10-15 mL / min (e.g., 10 mL / min, 11 mL / min, 12 mL / min, 13 mL / min, 14 mL / min, 15 mL / min, etc.).

[0028] Thirdly, the present invention provides the application of nucleic acid-lipid nanoparticles as described in the first aspect in the preparation of apolipoprotein E binding agents.

[0029] Fourthly, the present invention provides the use of nucleic acid-lipid nanoparticles as described in the first aspect in the preparation of medicaments for treating liver or kidney diseases.

[0030] Compared with the prior art, the present invention has the following beneficial effects: This invention creatively designs a lipid nanoparticle delivery system in which dimyristoyl phosphatidylcholine, ionizable lipids, cholesterol, and triglycerides synergistically work together. This allows the lipid nanoparticles of this invention to efficiently load nucleic acid drugs and actively target the liver or kidneys for efficient delivery, even without polyethylene glycol modification. Based on the crucial role of apolipoprotein E (ApoE) in liver-specific recognition and endocytosis, this invention enhances and predictably regulates liver delivery efficiency by modulating the interaction between the molecular structure of helper phospholipids in the lipid nanoparticles and ApoE. This provides a general and scalable design strategy for liver-targeted delivery of nucleic acid drugs. Attached Figure Description

[0031] Figure 1 Particle size, polymer dispersibility index (PDI), and zeta potential of nucleic acid-lipid nanoparticles in Example 1 and Comparative Examples 1-2 are shown. Figure 2 Transmission electron microscopy images of nucleic acid-lipid nanoparticles in Example 1 and Comparative Examples 1-2; Figure 3 The protein bands and semi-quantitative analysis diagrams for the gene silencing experiments of nucleic acid-lipid nanoparticles in Examples 1-4 are shown. Figure 4 Analysis of the micro-thermophoretic dynamics results of nucleic acid-lipid nanoparticles in Example 1 and Comparative Examples 1-2; Figure 5 The in vivo distribution analysis diagrams of nucleic acid-lipid nanoparticles in Example 1 and Comparative Examples 1-2 are shown. Figure 6 The graph shows the in vitro uptake of nucleic acid-lipid nanoparticles in Example 1 and Comparative Examples 1-2. Detailed Implementation

[0032] To further illustrate the technical means and effects of the present invention, the technical solution of the present invention will be further described below in conjunction with the accompanying drawings and preferred embodiments of the present invention. However, the present invention is not limited to the scope of the embodiments.

[0033] Example 1 This embodiment provides a nucleic acid-lipid nanoparticle, prepared by the following method: (1) Weigh each component according to the molar percentages of 8.6% DMPC, 19% DLin-MC3-DMA, 20.6% cholesterol and 51.8% tricaprylic acid glyceride, dissolve them in anhydrous ethanol to make the total lipid concentration 10 mg / mL, and form a homogeneous lipid mixture. With a nitrogen-to-phosphorus ratio of 6, the nucleic acid sequences shown in SEQ ID No. 1-SEQ ID No. 3 were mixed at a mass ratio of 1:1:1 to form the siRNA drug. The mixture was dissolved in a 10 mM citrate buffer (pH 4.0) and the final concentration of the siRNA drug was adjusted to 0.1 mg / mL to obtain the aqueous phase. Using a microfluidic chip, lipids and siRNA solution were rapidly mixed at a 3:1 (ethanol:buffer) flow rate of 12 mL / min. The mixture was then centrifuged at 5500 rpm for 10 min using a 100 kDa MWCO filter at 4°C. This process was repeated three times, with each resuspending in PBS (pH 7.4) to a final volume of 1 mL. The solution was stored at 4°C for further use.

[0034] Example 2 This embodiment provides a nucleic acid-lipid nanoparticle. The preparation method differs from that in Example 1 only in that the siRNA drug is replaced with a single sequence shown in SEQ ID No. 1, while all other conditions remain unchanged.

[0035] Example 3 This embodiment provides a nucleic acid-lipid nanoparticle. The preparation method differs from that in Example 1 only in that the siRNA drug is replaced with a single sequence shown in SEQ ID No. 2, while all other conditions remain unchanged.

[0036] Example 4 This embodiment provides a nucleic acid-lipid nanoparticle. The preparation method differs from that in Example 1 only in that the siRNA drug is replaced with a single sequence shown in SEQ ID No. 3, while all other conditions remain unchanged.

[0037] Comparative Example 1 This comparative example provides a nucleic acid-lipid nanoparticle. The preparation method differs from that of Example 1 only in that the equimolar amount of DMPC is replaced with DPPC, while all other conditions remain unchanged.

[0038] Comparative Example 2 This comparative example provides a nucleic acid-lipid nanoparticle. The preparation method differs from that of Example 1 only in that the equimolar amount of DMPC is replaced with DSPC, while all other conditions remain unchanged.

[0039] Test Example 1 This test case characterizes the nucleic acid-lipid nanoparticles (LNPs) of Example 1 and Comparative Examples 1-2.

[0040] DLS characterization: The obtained nucleic acid-lipid nanoparticles were diluted 10-fold with ultrapure water and analyzed by dynamic light scattering on a Malvern Zetasizer Nano ZS analyzer to determine the LNP particle size, polymer dispersibility index (PDI), and zeta potential. Results are as follows: Figure 1 As shown, the results indicate that the particle size of the three LNPs is in the range of 145-155 nm, and they have good dispersibility.

[0041] TEM characterization: 10 μL of LNPs was dropped onto a 200-mesh copper grid for 15 min. Excess liquid was absorbed with filter paper, followed by the addition of 5 μL of 3% uranium acetate dye for 1 min. Excess dye was then absorbed, and the morphology and structure of the LNPs were observed under a transmission electron microscope. The results are as follows: Figure 2 As shown in the figure, the results indicate that the three LNPs have good morphology.

[0042] Test Example 2 This test case demonstrates gene silencing experiments using the nucleic acid-lipid nanoparticles from Examples 1-4.

[0043] To screen for gene sequences that effectively silence C3, the nucleic acid-lipid nanoparticles from Examples 1-4 were named C3-123 (a mixture of three sequences), C3-1, C3-2, and C3-3, respectively (all three gene sequences were from Guangzhou Ruibo Company).

[0044] Western blot was used to investigate the in vitro silencing efficiency. AML12 cells in logarithmic growth phase were injected at a rate of 2 × 10⁻⁶ cells / mL. 5 Cells were seeded at a density of cells / well in 6-well cell culture plates and cultured at 37°C for 12 h. After culture, cells were transfected with C3 siRNAs containing different sequences encapsulated by Lipo2000 for 24 h. The control group was not treated. After transfection, the culture medium was discarded, and the cells were washed three times with PBS (pH 7.4) and collected. Then, Ripa lysis buffer containing protease inhibitors was added to the cells, the cells were shaken and lysed on ice for 15 min, and the cells were centrifuged (4°C, 14000g, 15 min) and the supernatant was collected. The protein content of each well was determined using a BCA protein assay kit. Then, the same concentration of protein was added to a precast gel for SDS-PAGE electrophoresis (80V 30 min-120V 60 min). After electrophoresis, the protein was transferred to a PVDF membrane for electroporation (250mA, 2 h). After electroporation, the membrane was blocked with 5% skim milk powder for 1 h. The membrane was then incubated with C3 antibody overnight at 4°C. After incubation, the membrane was incubated with secondary antibody for 1 h (25°C), and the protein bands were imaged using an imaging system.

[0045] The results are as follows Figure 3 As shown, both the protein banding results and quantitative analysis indicate that the knockout effect obtained by mixing the three sequences in Example 1 was the best.

[0046] Test Example 3 This application presents a micro-thermophoretic experiment on the nucleic acid-lipid nanoparticles of Example 1 and Comparative Examples 1-2.

[0047] Apolipoprotein APOE was dissolved in PBS (pH 7.4). The protein (target molecule) and dye molecules were co-incubated for 30 min to fluoresce the protein. The labeled protein was then purified using a desalting column. LNP ligand molecules were diluted 2-fold (totaling 16 concentrations) and co-incubated with the labeled protein for 15 min. The mixture of LNP ligand molecules and protein was loaded via capillary tube and then detected using micro-thermophoresis (MST). The binding affinity between the protein and the small molecule compound was analyzed using specialized software.

[0048] The results are as follows Figure 4As shown, LNP (Example 1) with DMPC as the auxiliary lipid exhibited the strongest binding affinity for ApoE (Kd = 1.69 μM), which was superior to the DSPC group (Kd = 9.52 μM) and the DPPC group (Kd = 30.44 μM). The experimental results indicate that by regulating the molecular structure of the auxiliary lipids in the lipid nanoparticles, the interaction ability of the lipid nanoparticles described in this invention with endogenous apolipoproteins in the blood during in vivo circulation was significantly enhanced, and they further exhibited a higher level of enrichment in liver tissue.

[0049] Test Example 4 This test case demonstrates the in vivo distribution of nucleic acid-lipid nanoparticles from Example 1 and Comparative Examples 1-2.

[0050] Using Cy5-siRNA as a fluorescent probe, eLNP(DMPC)@Cy5, eLNP(DPPC)@Cy5, and eLNP(DSPC)@Cy5 pre-incubated with ApoE protein, as well as LNP(DMPC)@Cy5, LNP(DPPC)@Cy5, and LNP(DSPC)@Cy5 without APOE incubation, were prepared using microfluidic technology. The distribution of these lipid nanoparticles in healthy C57 BL / 6 mice was then investigated.

[0051] The results are as follows Figure 5 As shown in the figure, the enrichment of LNPs (eLNPs) pre-incubated with APOE in the liver is illustrated. The liver targeting strength is in the order of eLNP (DMPC) > eLNP (DSPC) > eLNP (DPPC) > LNP (DMPC) > LNP (DSPC) > LNP (DPPC), which is consistent with the MST test results. DMPC has the strongest binding ability to APOE, resulting in the strongest liver targeting. The ApoE-LNP complex is recognized by the highly expressed LDL receptor on the surface of hepatocytes, which facilitates LNP entry into hepatocytes via endocytosis (such as clathrin-mediated endocytosis).

[0052] Test Example 5 This test case demonstrates the in vitro uptake of nucleic acid-lipid nanoparticles from Example 1 and Comparative Examples 1-2.

[0053] Using Cy5-siRNA as a fluorescent probe, eLNP(DMPC)@Cy5, eLNP(DPPC)@Cy5, eLNP(DSPC)@Cy5 pre-incubated with APOE protein, as well as LNP(DMPC)@Cy5, LNP(DPPC)@Cy5, and LNP(DSPC)@Cy5 without APOE incubation were prepared using microfluidic technology. The uptake of these lipid nanoparticles in normal mouse hepatocytes AML12 was then investigated.

[0054] The results are as follows Figure 6 As shown in the figure, pre-incubated APOE resulted in the strongest LNP (DMPC) uptake and the strongest binding affinity between DMPC and APOE. The LDL receptor, highly expressed on the surface of hepatocytes, recognizes the ApoE-LNP complex, mediated by the endocytosis pathway that allows LNP to enter hepatocytes.

Claims

1. A nucleic acid-lipid nanoparticle that enhances liver and kidney targeting, characterized in that, The nucleic acid-lipid nanoparticles include lipid nanoparticles and nucleic acid drugs encapsulated therein; The raw materials for preparing the lipid nanoparticles include myristoyl phosphatidylcholine, ionizable lipids, cholesterol, and triglycerides. The nucleic acid drug is an siRNA drug.

2. The nucleic acid-lipid nanoparticles according to claim 1, characterized in that, The molar percentages of each component in the raw materials for preparing the lipid nanoparticles are: 5%-10% myristoyl phosphatidylcholine, 15%-25% ionizable lipids, 15%-25% cholesterol, and 45%-60% triglycerides.

3. The nucleic acid-lipid nanoparticles according to claim 1 or 2, characterized in that, The ionizable lipids include any one or a combination of at least two of DLin-MC3-DMA, DLin-DMA, and DLin-KC2-DMA. Preferably, the ionizable lipid is DLin-MC3-DMA; Preferably, the triglyceride includes any one or a combination of at least two of tricaprylic acid glyceride, tricaprylic acid glyceride, and triheptanoic acid glyceride; Preferably, the triglyceride is trioctanoic acid glyceride.

4. The nucleic acid-lipid nanoparticles according to any one of claims 1-3, characterized in that, The siRNA drug targets the sequence of the complement component C3 gene; Preferably, the sequence of the siRNA drug is selected from any one or a combination of at least two of the following sequences: (1) The amino acid sequence shown in SEQ ID No. 1-SEQ ID No. 3, or, An amino acid sequence obtained by substituting, deleting, or adding 1-3 amino acid residues to the sequence described in (1), and having the same or similar function to the sequence described in (1), or, (3) An amino acid sequence that has at least 95% sequence homology with the sequence described in (1) or (2) and has the same or similar function as the sequence described in (1); Preferably, the siRNA drug is a mixture of sequences SEQ ID No. 1-SEQ ID No.

3.

5. The nucleic acid-lipid nanoparticles according to any one of claims 1-4, characterized in that, The nitrogen-to-phosphorus ratio of the ionizable lipids and siRNA is (4-8):

1.

6. The nucleic acid-lipid nanoparticles according to any one of claims 1-5, characterized in that, The nucleic acid-lipid nanoparticles have a particle size of 140-155 nm.

7. The method for preparing nucleic acid-lipid nanoparticles according to any one of claims 1-6, characterized in that, The preparation method includes: (1) Mix dimyristoyl phosphatidylcholine, ionizable lipids, cholesterol, triglycerides and organic solvents to obtain a lipid phase; The siRNA was mixed with an acidic buffer to obtain the aqueous phase. (2) Using a microfluidic device, the lipid phase and the aqueous phase are mixed to obtain the nucleic acid-lipid nanoparticles.

8. The preparation method according to claim 7, characterized in that, The mixing temperatures in step (1) are each independently 20-30℃; Preferably, the organic solvent in step (1) includes any one or a combination of at least two of methanol, ethanol, isopropanol, and n-butanol; Preferably, the acidic buffer solution in step (1) includes any one or a combination of at least two of citrate buffer, acetate buffer, and succinate buffer; Preferably, the mixing temperature in step (2) is 20-30°C; Preferably, the volumetric flow rate of the lipid phase and aqueous phase mixture in step (2) is (2-5):1, and the total flow rate is 10-15 mL / min.

9. The use of the nucleic acid-lipid nanoparticles according to any one of claims 1-6 in the preparation of apolipoprotein E binders.

10. The use of the nucleic acid-lipid nanoparticles according to any one of claims 1-6 in the preparation of medicaments for the treatment and / or prevention of liver or kidney diseases.

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