Nucleic acid lipid nanocarrier, preparation method therefor, and use thereof

Abstract

A nucleic acid lipid nanocarrier, a preparation method therefor, and use thereof. First, provided is a nanocarrier composition for a nucleic acid drug. On the basis that the total molar amount of lipids in the composition is 100%, the composition comprises the following lipid components: greater than or equal to 40% and less than or equal to 50% of ionizable lipid; greater than or equal to 0.5% and less than or equal to 3.0% of PEG-modified lipid; greater than or equal to 30% and less than or equal to 44.5% of steroid; and greater than 15% and less than 22% of helper phospholipid. Also provided are use of the composition in the preparation of a nucleic acid drug nanocarrier and the prepared nucleic acid-loaded drug. The nucleic acid drug nanocarrier can target a treatment site, and the non-liver-targeting lipid nanocarrier has low liver metastasis.

Description

A nucleic acid lipid nanocarrier, its preparation method and application Technical Field

[0001] This invention relates to a nucleic acid lipid nanocarrier, specifically to a lipid nanocarrier composition that can target therapeutic sites, a nucleic acid-loaded lipid nanocarrier, and their preparation methods and applications. Background Technology

[0002] Nucleic acid molecules are the foundation of life activities, and nucleic acid drugs work by introducing exogenous genes into target cells or tissues to replace, compensate for, block, or modify specific genes, thereby achieving the purpose of treating and preventing diseases. The research and development and production processes of nucleic acid drugs are relatively simple, with advantages such as short development cycles, high clinical development success rates, and good modifiability. However, ordinary nucleic acid molecules typically have a short circulation time in vivo and are easily degraded. Therefore, improving the in vivo delivery efficiency of nucleic acid drugs is one of the key research directions for improving the effectiveness of this type of product.

[0003] Currently, the most widely used delivery carrier for nucleic acid drugs is a multi-lipid nanoparticle. It possesses characteristics such as enhancing the efficacy of gene therapy and targeted delivery, protecting nucleic acids from rapid degradation in vivo, prolonging circulation time, and strengthening targeted delivery. Lipid nanoparticles (LNPs) typically consist of one ionizable lipid, one neutral phospholipid, one sterol (such as cholesterol), and one PEG lipid, with a zeta potential typically neutral (-15 to +15 mV) (Nano Lett. 2017, 17, 1326-1335). In preparation, an ethanol solution containing lipids is mixed with an aqueous buffer solution containing nucleic acids, and the ethanol is removed by dialysis or other methods, ultimately yielding nucleic acid-loaded LNPs (mRNA-LNPs).

[0004] The lipid nanoparticles of the classic formulation (US8058069B2, ionizable lipids: 50-60%; neutral lipids: 4-10%; cholesterol: 30-40%; PEG lipids: 0.5-2%) are mostly rapidly metabolized and excreted by the kidneys during circulation in the body. A portion accumulates in the liver via the bloodstream, and a smaller portion is distributed in the spleen, lymph nodes, and other tissues. High liver accumulation is highly detrimental to some mRNA-LNP vaccines or drugs that require non-hepatic targeting, as it not only reduces drug accumulation in non-hepatic sites but also increases the burden on the liver.

[0005] Sago et al. (Nat Biomed Eng 6(2)(2022)157-167) reported that when LNPs were prepared in a ratio of 80:20 (cationic lipid 7C1: PEG lipid), the mRNA was mainly delivered to the lungs; when prepared in a ratio of 60:10:5:25 (cationic lipid 7C1: cholesterol: DSPC: PEG lipid), the mRNA was mainly delivered to the spleen. These examples illustrate that different organs can be targeted by adjusting the composition and ratio of lipids. However, the cationic lipids used in this article were polyamino lipids, and there are currently no cases of non-liver-targeting LNPs prepared with monoamino lipids that have been adjusted in a certain ratio. Rurik et al. (Science 375,91–96(2022)) reported that modifying the surface of LNPs with CD5 antibodies can increase the expression of LNPs in T cells, but according to mouse anatomical imaging results, most LNPs are still enriched in the liver.

[0006] The inventors' preliminary research in this case showed that, considering ionizable lipids in the range of 42% to 48%, higher dosages resulted in higher transfection efficiency, although the significance was slightly lower; therefore, the transfection efficiency was considered equivalent between 42% and 48%. Similarly, considering DMG-PEG2000 in the range of 1.6% to 2.8%, higher dosages resulted in higher transfection efficiency, although the significance was slightly lower; therefore, the transfection efficiency was considered equivalent between 1.6% and 2.8%. And considering DSPC in the range of 0% to 6%, lower dosages resulted in higher transfection efficiency, exhibiting a significant trend. Relevant experiments can be found in CN118236344A, the disclosure of which is incorporated herein by reference.

[0007] Therefore, the industry still needs to develop more lipid nanocarriers that can target therapeutic sites, especially those with low liver metastasis and non-liver-targeting lipid nanocarriers. Summary of the Invention

[0008] One object of the present invention is to provide a nucleic acid drug nanocarrier composition.

[0009] Another object of the present invention is to provide applications of the composition.

[0010] Another objective of this invention is to provide a nucleic acid drug nanocarrier.

[0011] Another object of the present invention is to provide a method for preparing the nucleic acid drug nanocarrier.

[0012] Another object of the present invention is to provide the application of the aforementioned nucleic acid drug nanocarrier.

[0013] On one hand, the present invention provides a nanocarrier composition for nucleic acid drugs, wherein the composition comprises the following lipid components, based on a total molar amount of lipids of 100% in the composition:

[0014] Ionizable lipids: ≥40%, ≤50%;

[0015] PEG-modified lipids: ≥0.5% and ≤3.0%;

[0016] Steroids: ≥30%, ≤44.5%;

[0017] Cofactor phospholipids: greater than 15% and less than 22%.

[0018] According to a specific embodiment of the present invention, the nanocarrier composition for nucleic acid drugs of the present invention, based on a total molar amount of lipids of 100% in the composition, comprises the following lipid components:

[0019] Ionizable lipids: ≥40%, ≤50%;

[0020] PEG-modified lipids: ≥1.0% and ≤2.5%;

[0021] Steroids: ≥30%, ≤40%;

[0022] Co-phospholipids: greater than 15% and less than or equal to 20%.

[0023] According to a specific embodiment of the present invention, in the nanocarrier composition for nucleic acid drugs of the present invention, the ionizable lipid is a lipid containing an ionizable amino group.

[0024] According to a specific embodiment of the present invention, in the nanocarrier composition for nucleic acid drugs of the present invention, the ionizable lipid is a compound with the structure shown in Formula I or a drug-acceptable salt, tautomer, or stereoisomer thereof:

[0025] in:

[0026] L1 and L2 are linking bonds or divalent linkers, each of which is independently selected from -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -O-, -S-, -SS-, -C(=O)S-, -SC(=O)-, -N(R8)C(=O)-, -C(=O)N(R8)-, -N(R8)C(=O)O-, -OC(=O)N(R8)-, -SC(=O)N(R8)-, -N(R8)C(=O)S-, -C(=S)-, -SC(=S)-, and -C(=S)S-, where R8 is H or Cl-C. 12 alkyl;

[0027] R2 and R3 are independently substituted or unsubstituted C1-C 18 Straight-chain alkylene;

[0028] R4, R5, R6, and R7 are independently hydrogen, or substituted or unsubstituted C1-C atoms. 30 Aliphatic hydrocarbon group, or -R9-L4-R 10 The R9 and R 10 Each occurrence is independent of whether it is substituted or unsubstituted C1-C. 18 Aliphatic hydrocarbon group, L4 is O, S, alkenyl or alkynyl;

[0029] R1 is H, -R 11 -OR 11 -R 11 -OH, -R 11 -OR 12 -R 11 -OC(=O)R 12 -R 11 -NHC(=O)-R 12 -R 11 -OCH3 or -R 11 -N(R 12 (R) 13 ); R 11 For C1-C 12 Straight-chain alkyl or branched alkyl, R 12 and R 13 Each independently represents H or Cl-C 12 Straight-chain alkyl, or, R 12 With R 13 The N atom it is attached to forms C3-C 10 Heterocyclic alkyl groups.

[0030] According to a specific embodiment of the present invention, in the nanocarrier composition for nucleic acid drugs of the present invention, in Formula I:

[0031] L1 and L2 are either linking bonds or divalent linking groups, and each divalent linking group is independently selected from any one of -C(=O)-, -OC(=O)-, -C(=O)O-, -O-, -S-, -C(=O)S-, -SC(=O)-, -NHC(=O)-, and -C(=O)NH-.

[0032] R2 and R3 are independently substituted or unsubstituted C3-C9 straight-chain alkylene groups;

[0033] R4, R5, R6, and R7 are independently hydrogen, or substituted or unsubstituted C1-C atoms. 12 Aliphatic hydrocarbon group, or -R9-L4-R 10 The R9 and R 10 Each occurrence is independent of whether it is substituted or unsubstituted C1-C. 10 Aliphatic hydrocarbon group, L4 is O, S, alkenyl or alkynyl;

[0034] R1 is H, -R 11 or -R 11 -OH, where R 11 It is a C1-C6 straight-chain alkyl group.

[0035] According to a specific embodiment of the present invention, in the nanocarrier composition for nucleic acid drugs of the present invention, the ionizable lipid is selected from SM-102, Heptadecan-9-yl 8-((2-hydroxyethyl)(5-((2-methyl-3-(octylthio)propanoyl)oxy)pentyl)amino)octanoate, Heptadecan-9-yl 8-((7-((3-(hexylthio)-2-methylpropanoyl)oxy)heptyl)(2-hydroxyethyl)amino)octanoate, Heptadecan-9-yl 8-((2-hydroxyethyl)(5-((2-methyl-3-(octyloxy)propanoyl)oxy)pentyl)amino)octanoate, 7-((7-((3-(hexyloxy)-2-methylpropanoyl)oxy)heptyl)(2-hydroxyethyl)amino)heptyl 2-octyldecanoate, 6-((6-((2-hexyldecanoyl)thio)hexyl)(4-hydroxybutyl)amino)hexyl 2-hexyldecanoate, Heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecylthio)hexyl)amino)octanoate, or one or more of their tautomers and stereoisomers.

[0036] According to some specific embodiments of the present invention, the nanocarrier composition for nucleic acid drugs of the present invention, based on the total molar amount of lipids in the composition as 100%, wherein the amount of ionizable lipids is 42%-48%, preferably 45%-48%.

[0037] According to a specific embodiment of the present invention, in the nanocarrier composition for nucleic acid drugs of the present invention, the PEG-modified lipid is selected from one or more of DMG-PEG, DSPE-PEG, DMPE-PEG, DPPE-PEG, PEG-cer, DAG-PEG, and DMA-PEG.

[0038] According to a specific embodiment of the present invention, in the nanocarrier composition for nucleic acid drugs of the present invention, the PEG portion (e.g., mPEG-NH2) in the PEG-modified lipid has a size of 1000-5000 Da.

[0039] According to a specific embodiment of the present invention, in the nanocarrier composition for nucleic acid drugs of the present invention, the lipid portion of the PEG lipid has a length of C. 14 To C 18 .

[0040] According to some specific embodiments of the present invention, the nanocarrier composition for nucleic acid drugs of the present invention, based on the total molar amount of lipids in the composition being 100%, wherein the amount of PEG-modified lipids is 1.2%-2.2%, preferably 1.5%-2.2%.

[0041] According to a specific embodiment of the present invention, in the nanocarrier composition for nucleic acid drugs of the present invention, the steroid is cholesterol.

[0042] According to some specific embodiments of the present invention, the nanocarrier composition for nucleic acid drugs of the present invention, based on the total molar amount of lipids in the composition as 100%, contains 32%-40% steroids, preferably 32.5%-38%, and more preferably 33.5%-37%.

[0043] According to a specific embodiment of the present invention, in the nanocarrier composition for nucleic acid drugs of the present invention, the auxiliary phospholipid is selected from one or more of DSPC, DPPC, DMPC, DOPC, DOPS, POPC or DOPE, preferably DSPC or DOPE.

[0044] According to some specific embodiments of the present invention, the nanocarrier composition for nucleic acid drugs of the present invention, based on the total molar amount of lipids in the composition being 100%, wherein the amount of auxiliary phospholipids is 15.2%-19%, preferably 15.5%-18%, and more preferably 16%-17%.

[0045] On the other hand, the present invention also provides the application of the composition in the preparation of nucleic acid drug nanocarriers.

[0046] On the other hand, the present invention also provides a nucleic acid drug nanocarrier, which is prepared by loading a nucleic acid drug onto the composition described in the present invention.

[0047] According to a specific embodiment of the present invention, the nucleic acid drug nanocarrier of the present invention has a particle size of 50nm-200nm, preferably 60nm-160nm; an encapsulation efficiency of greater than 80%; a potential of -15mV to +15mV, preferably -10mV to +10mV; and a pKa of 6.0-7.2, preferably 6.2-7.0.

[0048] According to a specific embodiment of the present invention, the nucleic acid in the nucleic acid drug nanocarrier of the present invention is mRNA.

[0049] According to a specific embodiment of the present invention, the nucleic acid in the nucleic acid drug nanocarrier of the present invention comprises 100 to 50,000 nucleotides, or 200 to 20,000 nucleotides, or 300 to 10,000 nucleotides, or 500 to 9,000 nucleotides.

[0050] According to a specific embodiment of the present invention, in the nucleic acid drug nanocarrier of the present invention, the nitrogen-to-phosphorus ratio of nucleic acid to ionizable lipid is 3-10, preferably 4-8, and more preferably 5-7.

[0051] On the other hand, the present invention also provides a method for preparing nucleic acid drug nanocarriers, the method comprising:

[0052] An ethanol solution containing the lipid composition described in this invention is prepared and mixed with an aqueous buffer solution containing a nucleic acid drug to obtain a nanocarrier loaded with the nucleic acid drug.

[0053] According to a specific embodiment of the present invention, in the preparation method of the nucleic acid drug nanocarrier of the present invention, the pH of the aqueous buffer solution is 3-7, preferably 3-5, and more preferably 3.5-5.

[0054] According to a specific embodiment of the present invention, in the preparation method of the nucleic acid drug nanocarrier of the present invention, the mixing of an ethanol solution containing a lipid composition with an aqueous buffer solution containing a nucleic acid drug is performed using a microfluidic chip.

[0055] According to a specific embodiment of the present invention, in the preparation method of the nucleic acid drug nanocarrier of the present invention, the nitrogen-to-phosphorus ratio of nucleic acid to ionizable lipid in the mixed system obtained after mixing is 3-10:1; preferably 4-8:1, and more preferably 5-6.5:1.

[0056] According to a specific embodiment of the present invention, in the preparation method of the nucleic acid drug nanocarrier of the present invention, the mixed system obtained after mixing is subjected to subsequent processing; the subsequent processing includes replacing the ethanol in the buffer solution with one or more combinations of water, NaCl, PBS, HEPES, PB, Tris, citric acid, and acetate buffer; wherein, the pH of the solution system after replacement is 4.5 to 8, preferably 6 to 8.

[0057] According to a specific embodiment of the present invention, the prepared nanocarrier carrying nucleic acid drugs is stored at 2 to 25°C, or stored at -80 to -20°C after adding a cryoprotectant, or stored after lyophilization treatment after adding a lyophilization protectant.

[0058] According to a specific embodiment of the present invention, in the preparation method of the nucleic acid drug nanocarrier of the present invention, the cryoprotectant and lyophilization protectant are each independently selected from one or more of sucrose, trehalose, mannitol, mannose, glucose, β-cyclodextrin, and hyaluronic acid.

[0059] On the other hand, the present invention also provides the application of the nucleic acid drug nanocarrier described above or the nucleic acid drug nanocarrier prepared by the method in the preparation of nucleic acid drugs.

[0060] According to some specific embodiments of the present invention, the nucleic acid drug nanocarrier of the present invention has a low liver metastasis in the target organism and is a non-liver-targeting lipid nanocarrier.

[0061] According to a specific embodiment of the present invention, the nucleic acid drug of the present invention is used to target and induce protein expression in subjects.

[0062] According to some specific embodiments of the present invention, the nucleic acid drug of the present invention is an intravenous injection formulation or an intramuscular injection formulation, which is used to deliver the nucleic acid drug into the immune cells of a subject.

[0063] According to some specific embodiments of the present invention, the nucleic acid drug of the present invention is an intramuscular injection formulation, which is used to deliver the nucleic acid drug to a non-liver site (e.g., spleen) of the subject.

[0064] According to some specific embodiments of the present invention, the nucleic acid drug of the present invention is an intra-articular injection formulation, which is used to deliver the nucleic acid drug to the joint site of the subject. The joint may be, for example, the knee joint, elbow joint, etc.

[0065] According to some specific embodiments of the present invention, the nucleic acid drug of the present invention is a tumor tissue injection formulation, which is used to deliver the nucleic acid drug to the tumor site of a subject. Preferably, the tumor is a solid tumor, such as a subcutaneous tumor.

[0066] According to a specific embodiment of the present invention, the nucleic acid drug of the present invention is non-liver-targeting. This nucleic acid drug has low liver metastasis and can be used to induce protein expression in non-liver sites in subjects.

[0067] According to some specific embodiments of the present invention, the subject of the nucleic acid drug nanocarrier is a mammal or a human, preferably a primate.

[0068] In summary, this invention provides a nucleic acid lipid nanocarrier that can target therapeutic sites, its preparation method, and its application, wherein the non-liver-targeting lipid nanocarrier has low liver metastasis. Attached Figure Description

[0069] Figure 1 is a fluorescence imaging image of rats after intra-articular injection in Example 3 of the present invention (*=p<0.05, **=p<0.01, ***=p<0.001, one-way ANOVA test).

[0070] Figure 2 shows the fluorescence imaging of mice after in situ injection into the tumor in Example 4 of this invention (* = p < 0.05, ** = p < 0.01, *** = p < 0.001, one-way ANOVA test).

[0071] Figure 3 shows the results of model fitting analysis using standard least squares method and effect screening on liver imaging data after intramuscular injection in the thigh of mice in Example 5 of this invention.

[0072] Figure 4 shows the results of model fitting analysis using standard least squares method and effect screening on spleen imaging data after intramuscular injection in the thigh of mice in Example 5 of this invention.

[0073] Figure 5 shows the evaluation results of the mRNA-LNP immune cell targeting effect in Example 6 of the present invention.

[0074] Figure 6 shows the evaluation results of the mRNA-LNP immune effect in Example 7 of the present invention. Detailed Implementation

[0075] To provide a clearer understanding of the technical features, objectives, and beneficial effects of this invention, the technical solution of this invention will now be described in detail with reference to specific embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of application of this invention. In the embodiments, all original reagent materials are commercially available, and experimental methods not specifically specified are conventional methods and conditions well known in the art, or according to the conditions recommended by the instrument manufacturer.

[0076] Unless otherwise specifically defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Every reference mentioned or cited herein is incorporated in its entirety through citation.

[0077] Example 1: mRNA-LNP formulation design, preparation and characterization

[0078] Heptadecan-9-yl 8-((2-hydroxyethyl)(5-((2-methyl-3-(octylthio)propanoyl)oxy)pentyl)amino)octanoate (35# ionizable lipid, also referred to as compound 35 in this invention, which accounts for 48% of LNP), DSPC (0-25%), cholesterol (25%-49.8%), and DMG-PEG2000 (1.5%-2.2%) were dissolved in ethanol according to the molar ratios shown in Table 1, with a total concentration of 10 mg / mL. mRNA (approximately 2000 nucleotides in length for experimental use) was diluted in 100 mM citrate-sodium citrate buffer (pH 4). The final mRNA concentration was based on a nitrogen-to-phosphorus ratio of 6:1 to the ionizable lipid.

[0079] 3 mL of mRNA buffer and 1 mL of lipid solution were respectively loaded into two 5 mL syringes, which were then attached to a microfluidic injection pump. The chip was connected to the syringe, the injection pump flow rate was set, and the start button on the injection pump was pressed to inject the chip at a flow rate ratio of 3:1. The color of the product exiting the chip was observed. The first 5 milky white droplets (approximately 100 μL) were discarded, and the remaining sample was collected into an EP tube. The collected product was placed in a dialysis bag and dialyzed against 10 mM PBS (pH 7.4) for 6 hours (molecular weight cutoff: 100 kDa). Subsequently, the product was ultrafiltered to the ideal concentration, and the lipid nanoparticles were filtered through a 0.22 μm sterile filter and stored at 4 °C.

[0080] Following the Ribogreen kit instructions, the encapsulation efficiency of the product was calculated; particle size and polydispersity index (PDI) were determined, and zeta potential was analyzed using standard detection methods on a Malvern Zetasizer nano instrument.

[0081] The percentage content of each component in the lipid composition, the particle size, PDI and encapsulation efficiency of the obtained mRNA-loaded LNPs are shown in Table 1.

[0082] Table 1. Characterization results of nanoparticles prepared with different formulations

[0083] The results showed that the nanoparticles No. 1 to No. 6 prepared in this embodiment had high encapsulation efficiency and uniform particle size, which met the basic characteristics of nucleic acid delivery vectors; the encapsulation efficiency of nanoparticle No. 7 was slightly lower, which may be due to the excessive DSPC content.

[0084] Example 2: Determination of the in vivo expression effect of luciferase mRNA delivered via tail vein using nanolipid particles.

[0085] BALB / c mice aged 6-8 weeks were injected via tail vein with LUC-mRNA-lipid nanoparticles containing 5 μg of mRNA (the nucleotide sequence of LUC-mRNA is shown in SEQ ID NO:1 of CN114380724B), prepared using the same method as in Example 1. The following day, mice were injected intraperitoneally with 100 μg of D-Luciferin Potassium Salt, and the results were detected using a PerkinElmer small animal imaging system. Fluc is commonly used in mammalian cell cultures to measure gene expression and cell viability; it emits biofluorescence in the presence of its substrate, luciferin. The mRNA used was characterized by an ARCA cap structure, a polyA tail length of 100-120 nt, and complete substitution of pseudouracil. The percentage content of each component in the lipid composition is shown in Table 1. After in vivo imaging, the mice were dissected, and the heart, liver, spleen, lung, and kidney were harvested for ex vivo organ imaging. The fluorescence intensity at the heart, lung, and kidney was close to the background value, thus excluding the possibility of large amounts of LNP enrichment and expression in these organs. Fluorescence imaging data are shown in Table 2.

[0086] Table 2. Fluorescence imaging results of mouse nanoparticles prepared with different formulations

[0087] Table 2 shows the in vivo imaging results for each group. For DMG-PEG2000, the in vivo transfection efficiency was comparable when the concentration ranged from 1.5% to 2.2%. For DSPC, the lower concentration resulted in higher transfection efficiency in the liver, while the higher concentration resulted in higher transfection efficiency in the spleen, exhibiting a significant correlation. When the DSPC content exceeded 20%, the nanoparticle encapsulation rate decreased, drug-likeness declined, and the overall brightness of the liver and spleen decreased compared to 20% DSPC.

[0088] Example 3: Expression and distribution of mRNA-LNP after intra-articular injection into the knee joint

[0089] Novel lipid nanoparticles (mRNA-LNPs) encapsulating luciferase mRNA were prepared using Heptadecan-9-yl 8-((7-((3-(hexylthio)-2-methylpropanoyl)oxy)heptyl)(2-hydroxyethyl)amino)octanoate (51# ionizable lipid, also referred to as compound 51 in this invention), following the method described in Example 1. After preparation, the sample was concentrated to 0.3 mg / ml. SD rats were injected with 15 μg / 50 μl of naked luciferase mRNA or mRNA-LNP into each knee joint. 24 h later, the rats were intraperitoneally injected with 200 μg of D-Luciferin Potassium Salt, and fluorescence imaging was performed. The percentage content of each component in the lipid composition and the detection results are shown in Table 3. The fluorescence imaging is shown in Figure 1.

[0090] Table 3. Physicochemical characterization results of nanoparticles prepared with different formulations

[0091] The results of this embodiment show that mRNA-LNP expression at the knee joint is generally higher than that of naked mRNA. Furthermore, with higher DSPC levels, there is less liver metastasis of mRNA-LNP, and the in-situ expression level is higher after intra-articular injection into the knee joint. This type of formulation is suitable for some nucleic acid drugs requiring high intra-articular expression in the knee joint, such as the expression of chondrocyte regeneration factor used in the treatment of osteoarthritis. Moreover, due to lower liver metastasis, hepatotoxicity is also lower.

[0092] Example 4: mRNA-LNP expression and distribution at tumor sites after injection

[0093] Following the method described in Example 1, lipid nanoparticles (mRNA-LNPs) encapsulating luciferase mRNA were prepared using heptadecan-9-yl 8-((2-hydroxyethyl)(5-((2-methyl-3-(octyloxy)propanoyl)oxy)pentyl)amino)octanoate (hereinafter referred to as lipid 7), 7-((7-((3-(hexyloxy)-2-methylpropanoyl)oxy)heptyl)(2-hydroxyethyl)amino)heptyl 2-octyldecanoate (hereinafter referred to as lipid 17), SM-102 (hereinafter referred to as lipid SM), and ALC-0315 (hereinafter referred to as lipid A5). The physicochemical properties of each mRNA-LNP are shown in Table 4. Six- to eight-week-old female C57BL6 mice were used. The mice were housed in individually ventilated cages with constant temperature and humidity, under 12-hour light exposure, at a room temperature of 23-25°C and a humidity of 30-60%. The mice were fed with 5 × 10⁻⁶... 5 One B16F10 cell per mouse was subcutaneously injected into the right abdomen. Tumor growth was observed and recorded regularly. On day 8 post-inoculation, the length (L, mm) and width (D, mm) of the tumor were measured using calipers, and the tumor volume (V) was calculated using the formula: V = (L × D) / (L × D) * ... 2 ) / 2, take tumor volume between 80-120mm 3 Mice were randomly divided into groups and injected intratumorally with 4 μg of lipid nanoparticles of different types carrying luciferase mRNA. After 24 hours, the liver, spleen and tumor were dissected for imaging.

[0094] Table 4. Physicochemical characterization results of nanoparticles prepared with different formulations

[0095] The fluorescence imaging of tumor expression distribution after in situ injection in this embodiment is shown in Figure 2. The results show that as the dosage of DSPC increases, the amount of mRNA-LNP enriched in the liver decreases, while the amount retained within the tumor increases. This indicates that higher dosage formulations of DSPC are suitable for some nucleic acid drugs requiring high in situ expression within the tumor, such as the expression of some tumor treatment-related cytokines. Furthermore, due to lower liver metastasis, hepatotoxicity is also lower.

[0096] Example 5: Evaluation of in vivo delivery effect of intramuscular injection

[0097] This example validated the in vivo transfection efficiency of different ratios of SM-102, DOPE, DMG-PEG2K, and CHOL, as shown in Table 5. The preparation and characterization methods were the same as in Example 1. 5 μg of LUC-mRNA was injected intramuscularly into the thigh of each mouse. The liver and spleen were dissected and imaged the following day. Data were analyzed using JMP Pro 16.0 software, employing standard least squares and effect screening for model fitting. The analysis results are shown in Figure 3 (liver) and Figure 4 (spleen).

[0098] Table 5. Physicochemical characterization results of nanoparticles prepared with different formulations

[0099] As shown in Table 5 and Figures 3 and 4, SM-102 in the range of 40% to 50% and DMG-PEG2K in the range of 1.5% to 2.2% have no significant effect on transfection distribution. When DOPE is in the range of 5% to 16%, its distribution in the spleen gradually increases and its enrichment in the liver gradually decreases with increasing dosage, showing a significant trend.

[0100] Example 6: Evaluation of Immune Cell Targeting Effect

[0101] Delivery vectors with different lipid ratios were synthesized using GFP mRNA according to the method in Example 1.

[0102] After preparation, the sample was concentrated to 0.2 mg / ml. Balb / c mice were injected via tail vein with 20 μg / 100 μl PBS or mRNA-LNP. After 24 hours, the mice were dissected, and spleen cells were collected, separated, stained, and subjected to flow cytometry. The percentage content of each component in the lipid composition and the detection results are shown in Table 6 and Figure 5.

[0103] Table 6. Physicochemical characterization results of nanoparticles prepared with different formulations

[0104] The results show that the LNP 2 prepared by the formulation of this invention has a high transfection efficiency of immune cells (T cells, B cells, and DC cells). Combined with the results of Example 2, this suggests that the LNP may be well-suited for transfection targeting immune organs and immune cells with low hepatotoxicity.

[0105] Example 7: Evaluation of Immunological Effect

[0106] Balb / c mice aged 6-8 weeks were immunized by intramuscular injection on days 0 and 14 with mRNA vaccines for SARS-CoV-2 infection delivered by different nanolipid particle compositions (corresponding nucleotide sequences are shown in CN114380724B, SEQ ID NO:6), 2.5 μg / mouse. Blood samples were collected on day 21, and the binding antibody titer was measured by ELISA, and the cellular immunity level was measured by ICS to evaluate the protective efficacy of mRNA vaccines for SARS-CoV-2 infection delivered by different nanolipid particle compositions against SARS-CoV-2 virus infection. The results are shown in Table 7 and Figure 6.

[0107] Table 7. Physicochemical characterization results of nanoparticles prepared with different formulations

[0108] The results showed that with increasing DSPC dosage, humoral immunity levels decreased slightly, while cellular immunity levels gradually increased. The formulation of this invention is well-suited for disease prevention or therapeutic products that require high levels of cellular immunity.

Claims

1. A nanocarrier composition for a nucleic acid drug, comprising the following lipid components, based on the total moles of lipids in the composition being 100%: ionizable lipid: greater than or equal to 40% and less than or equal to 50%; PEG-modified lipid: greater than or equal to 0.5% and less than or equal to 3.0%; steroid: greater than or equal to 30% and less than or equal to 44.5%; co-phospholipid: greater than 15% and less than 22%.

2. The composition of claim 1, comprising the following lipid components, based on the total moles of lipids in the composition being 100%: ionizable lipid: greater than or equal to 40% and less than or equal to 50%; PEG-modified lipid: greater than or equal to 1.0% and less than or equal to 2.5%; steroid: greater than or equal to 30% and less than or equal to 40%; co-phospholipid: greater than 15% and less than or equal to 20%.

3. The composition according to claim 1 or 2, wherein, the ionizable lipid is a lipid containing one ionizable amine group; Further preferably, the ionizable lipid is a compound of the structure shown in Formula I or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof: wherein: L1, L2are a bond or a divalent linking group, each independently selected from any one of -C(=0)-, -OC(=0)-, -C(=0)0-, -OC(=0)0-, -0-, -S-, -S-S-, -C(=0)S-, -SC(=0)-, -N(R8)C(=0)-, -C(=0)N(R8)-, -N(R8)C(=0)0-, -OC(=0)N(R8)-, -SC(=0)N(R8)-, -N(R8)C(=0)S-, -C(=S)-, -SC(=S)-, and -C(=S)S-, said R8being H or C1-C6alkyl; 12 alkyl; R2and R3are independently substituted or unsubstituted C1-C 18 straight-chain alkylene; R4, R5, R6, R7are independently hydrogen, or a substituted or unsubstituted C1-C 30 a substituted or unsubstituted C1-C 10 R9, R 10 each occurrence is independently a substituted or unsubstituted C1-C 18 a substituted or unsubstituted C1-C R1is H, -R 11 , -OR 11 , -R 11 -OH, -R 11 -OR 12 , -R 11 -OC(=O)R 12 , -R 11 -NHC(=O)-R 12 , -R 11 -OCH3or -R 11 -N(R 12 )(R 13 ); R 11 is C1-C 12 straight or branched chain alkyl, R 12 and R 13 are each independently H or C1-C 12 straight chain alkyl, or, R 12 and R 13 and the N atom to which they are attached form a C3-C 10 heterocycloalkyl.

4. The composition of claim 3, wherein, in Formula I: L1, L2 are a linking group or a divalent linking group, each independently selected from any one of -C(=0)-, -OC(=0)-, -C(=0)0-, -0-, -S-, -C(=0)S-, -SC(=0)-, -NHC(=0)-, -C(=0)NH-; R2and R3are independently substituted or unsubstituted C3-C9 straight chain alkylene; R4, R5, R6, R7are independently hydrogen, or a substituted or unsubstituted C1-C 12 a substituted or unsubstituted aliphatic hydrocarbon group, or is -R9-L4-R 10 ; said R9, R 10 independently at each occurrence is a substituted or unsubstituted C1-C 10 a substituted or unsubstituted aliphatic hydrocarbon group, L4is O, S, alkenyl or alkynyl; R1is H, -R 11 or -R 11 -OH, wherein R 11 is a C1-C6 linear alkyl group.

5. The composition of claim 3, wherein, The ionizable lipid is selected from one or more of SM-102, Heptadecan-9-yl 8-((2-hydroxyethyl)(5-((2-methyl-3-(octylthio)propanoyl)oxy)pentyl)amino)octanoate, Heptadecan-9-yl 8-((7-((3-(hexylthio)-2-methylpropanoyl)oxy)heptyl)(2-hydroxyethyl)amino)octanoate, Heptadecan-9-yl 8-((2-hydroxyethyl)(5-((2-methyl-3-(octyloxy)propanoyl)oxy)pentyl)amino)octanoate, 7-((7-((3-(hexyloxy)-2-methylpropanoyl)oxy)heptyl)(2-hydroxyethyl)amino)heptyl 2-octyldecanoate, 6-((6-((2-hexyldecanoyl)thio)hexyl)(4-hydroxybutyl)amino)hexyl 2-hexyldecanoate, Heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecylthio)hexyl)amino)octanoate, or tautomers and stereoisomers thereof.

6. The composition according to any one of claims 1-5, wherein, The PEG-modified lipid is selected from one or more of DMG-PEG, DSPE-PEG, DMPE-PEG, DPPE-PEG, PEG-cer, DAG-PEG, DMA-PEG; Preferably, the size of the PEG moiety in the PEG-modified lipid is between 1000 and 5000 Da, and the length of the lipid moiety of the PEG-lipid is between C 14 to C 18 .

7. The composition according to any one of claims 1-6, wherein, The steroid is cholesterol.

8. The composition according to any one of claims 1-7, wherein, The helper phospholipid is selected from one or more of DSPC, DPPC, DMPC, DOPC, DOPS, POPC, or DOPE; preferably DSPC or DOPE.

9. Use of the composition of any one of claims 1-8 in the manufacture of a nucleic acid drug nanocarrier.

10. A nucleic acid drug nanocarrier prepared by loading a nucleic acid drug into the composition of any one of claims 1-9.

11. The nucleic acid drug nanocarrier of claim 10, having a particle size of 50-200 nm, preferably 60-160 nm; an encapsulation efficiency of greater than 80%; a zeta potential of -15 mV to +15 mV, preferably -10 mV to +10 mV; and a pKa of 6.0-7.2, preferably 6.2-7.

0.

12. The nucleic acid drug nanocarrier according to claim 10 or 11, wherein, The nucleic acid is mRNA. Preferably, the nucleic acid comprises 100 to 50000 nucleotides, or 200 to 20000 nucleotides, or 300 to 10000 nucleotides, or 500 to 9000 nucleotides.

13. The nucleic acid drug nanocarrier of any one of claims 10-12, wherein, The nucleic acid has a nitrogen to phosphorus ratio of 3 to 10, preferably 4 to 8, and more preferably 5 to 7, with the ionizable lipid.

14. A method for preparing a nucleic acid drug nanocarrier, the method comprising: Preparation of an ethanol solution containing the lipid composition of any one of claims 1-8, mixing with an aqueous buffer containing a nucleic acid drug, to obtain a nucleic acid drug-loaded nanocarrier.

15. Use of the nucleic acid drug nanocarrier of any one of claims 10-13 or the nucleic acid drug nanocarrier prepared by the method of claim 14 in the preparation of a nucleic acid drug.

16. Use according to claim 15, wherein, The nucleic acid drug is for targeted induction of protein expression in a subject.

17. The use of claim 15, wherein: The nucleic acid drug is in an intravenous injection dosage form or a muscle injection dosage form, and the nucleic acid drug is for delivering the nucleic acid drug in immune cells of the subject; or The nucleic acid drug is in a muscle injection dosage form, and the nucleic acid drug is for delivering the nucleic acid drug at a non-liver site (e.g., spleen) of the subject; or The nucleic acid drug is in an intra-articular injection dosage form, and the nucleic acid drug is for delivering the nucleic acid drug at a joint site of the subject; or The nucleic acid drug is in a tumor tissue injection dosage form, and the nucleic acid drug is for delivering the nucleic acid drug at a tumor site of the subject; preferably, the tumor is a solid tumor, e.g., a subcutaneous tumor.

18. The use according to any one of claims 15 to 17, wherein, The nucleic acid drug is non-liver targeted.

19. The use of any one of claims 15-18, wherein the subject is a mammal or a human, and the mammal is preferably a primate.

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