Nucleic acid lipid nano-carrier as well as preparation method and application thereof

Abstract

The invention provides a nucleic acid lipid nano-carrier as well as a preparation method and application thereof. The invention firstly provides a nano-carrier composition for a nucleic acid drug, and the nano-carrier composition comprises the following lipid components by taking the total molar weight of lipid in the composition as 100%: greater than or equal to 40% and less than or equal to 50% of ionizable lipid; the PEG modified lipid is greater than or equal to 0.5% and less than or equal to 3.0%; steroid: more than or equal to 30% and less than or equal to 44.5%; and more than 15% and less than 22% of auxiliary phospholipids. The invention also provides application of the composition in preparation of a nucleic acid drug nano-carrier and the prepared nucleic acid-loaded drug. The nucleic acid drug nano-carrier disclosed by the invention can target a treatment part, and a non-liver-targeted lipid nano-carrier has low liver metastasis.

Description

TECHNICAL FIELD

[0001] The present application relates to a nucleic acid lipid nanocarrier, in particular, to a lipid nanocarrier composition capable of targeting a treatment site, a nucleic acid-loaded lipid nanocarrier, and a preparation method and application thereof. BACKGROUND

[0002] Nucleic acid molecules are the basis of life activities, and nucleic acid drugs are used to introduce exogenous genes into target cells or tissues to replace, compensate, block or correct specific genes, so as to achieve the purpose of treating and preventing diseases. The research and development production process of nucleic acid drugs is relatively simple, and has the advantages of short research and development cycle, high success rate of clinical development, and good plasticity for improvement. However, ordinary nucleic acid molecules usually have a short circulation time in the body and are easily degraded. Therefore, improving the in vivo delivery efficiency of nucleic acid drugs is one of the key research directions to improve the effectiveness of such products.

[0003] At present, the most widely used delivery carrier for nucleic acid drugs is a nanoparticle composed of multiple lipid components, which has the characteristics of improving the efficacy of gene drugs and targeted delivery, etc., can protect nucleic acids from being rapidly degraded in the body, prolong the circulation time, and enhance targeted delivery. Lipid nanoparticles (LNP) are usually composed of one ionizable lipid, one neutral phospholipid, one steroid (such as cholesterol), and one PEG lipid, and the zeta potential is usually electrically neutral (-15~+15 mV) (Nano Lett. 2017, 17, 1326-1335). During preparation, an ethanol solution containing lipids and an aqueous buffer containing nucleic acids are mixed together, and then ethanol is removed by dialysis or other methods to obtain nucleic acid-loaded LNP (mRNA-LNP).

[0004] The classic formula (US8058069B2, ionizable lipid: 50-60%; neutral lipid: 4-10%; cholesterol: 30-40%; PEG lipid: 0.5-2%) of the lipid nanoparticle circulates in the body, most of which is rapidly metabolized and excreted out of the body through the kidneys, part of which is enriched in the liver with blood, and a small part is distributed in the spleen, lymph and other tissues. High liver enrichment is very unfavorable for some mRNA-LNP vaccines or drugs that require non-liver targeting, not only reducing drug enrichment in non-liver sites, but also increasing 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]

[0026] in:

[0027] 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;

[0028] R2 and R3 are independently substituted or unsubstituted C1-C 18 Straight-chain alkylene;

[0029] 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;

[0030] 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 ,

[0031] -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.

[0032] According to a specific embodiment of the present invention, in the nanocarrier composition for nucleic acid drugs of the present invention, in formula I:

[0033] 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-.

[0034] R2 and R3 are independently substituted or unsubstituted C3-C9 straight-chain alkylene groups;

[0035] R4, R5, R6, and R7 are independently hydrogen, or substituted or unsubstituted C1-C atoms. 12Aliphatic 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;

[0036] R1 is H, -R 11 or -R 11 -OH, where R 11 It is a C1-C6 straight-chain alkyl group.

[0037] 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-yl8-((2-hydroxyethyl)(5-((2-methyl-3-(octyloxy)propanoyl)oxy)pentyl)amino)octanoate,7-(( 7-((3-(hexyloxy)-2-methylpropanoyl)oxy)heptyl)(2-hydroxyethyl)amino)heptyl2-octyldecanoate, 6-((6-((2-hexyldecanoyl)thio)hexyl)(4-hydroxybutyl)amino)hexyl2-hexyldecanoate, Heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecylthio)hexyl)amino)octanoate or one or more of its tautomers and stereoisomers.

[0038] 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%.

[0039] 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.

[0040] 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.

[0041] 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 .

[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 being 100%, wherein the amount of PEG-modified lipids is 1.2%-2.2%, preferably 1.5%-2.2%.

[0043] 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.

[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 as 100%, contains 32%-40% steroids, preferably 32.5%-38%, and more preferably 33.5%-37%.

[0045] 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.

[0046] 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%.

[0047] On the other hand, the present invention also provides the application of the composition in the preparation of nucleic acid drug nanocarriers.

[0048] 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.

[0049] 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.

[0050] According to a specific embodiment of the present invention, the nucleic acid in the nucleic acid drug nanocarrier of the present invention is mRNA.

[0051] 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.

[0052] 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.

[0053] On the other hand, the present invention also provides a method for preparing nucleic acid drug nanocarriers, the method comprising:

[0054] 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.

[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 pH of the aqueous buffer solution is 3-7, preferably 3-5, and more preferably 3.5-5.

[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 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.

[0057] 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.

[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 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.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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

[0071] Figure 1 This 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).

[0072] Figure 2 This is a fluorescence imaging image of mice after in situ injection into the tumor in Example 4 of the present invention (*=p<0.05, **=p<0.01, ***=p<0.001, one-way ANOVA test).

[0073] Figure 3 This shows the results of model fitting analysis using standard least squares and effect screening on liver imaging data after intramuscular injection in the thigh of mice in Example 5 of this invention.

[0074] Figure 4 This invention demonstrates 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.

[0075] Figure 5 This shows the evaluation results of the mRNA-LNP immune cell targeting effect in Example 6 of the present invention.

[0076] Figure 6 This shows the evaluation results of the mRNA-LNP immune effect in Example 7 of the present invention. Detailed Implementation

[0077] 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.

[0078] Unless otherwise specifically defined, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art in the relevant field.

[0079] Example 1: mRNA-LNP formulation design, preparation and characterization

[0080] 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.

[0081] 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.

[0082] 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 Zetasizernano instrument.

[0083] 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.

[0084] Table 1. Characterization results of nanoparticles prepared with different formulations

[0085]

[0086] 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.

[0087] Example 2: Determination of the in vivo expression effect of luciferase mRNA delivered via tail vein using nanolipid particles.

[0088] 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.

[0089] Table 2. Fluorescence imaging results of mouse nanoparticles prepared with different formulations

[0090]

[0091]

[0092] 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.

[0093] Example 3: Expression and distribution of mRNA-LNP after intra-articular injection into the knee joint

[0094] 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). 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. Fluorescence imaging images are shown below. Figure 1 .

[0095] Table 3. Physicochemical characterization results of nanoparticles prepared with different formulations

[0096]

[0097] 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.

[0098] Example 4: mRNA-LNP expression and distribution at tumor sites after injection

[0099] 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)heptyl2-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 x 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.

[0100] Table 4. Physicochemical characterization results of nanoparticles prepared with different formulations

[0101]

[0102] The fluorescence imaging image of tumor orthotopic injection expression distribution in this embodiment is shown below. Figure 2 The results showed that with increasing DSPC dosage, the amount of mRNA-LNP metastasized and enriched in the liver decreased, while the amount retained and expressed within the tumor increased. This indicates that higher-dose DSPC formulations are suitable for some nucleic acid drugs requiring high in situ expression within the tumor, such as the expression of some tumor therapy-related cytokines. Furthermore, due to lower liver metastasis, hepatotoxicity is also lower.

[0103] Example 5: Evaluation of in vivo delivery effect of intramuscular injection

[0104] This example verified the in vivo transfection efficiency of different ratios of SM-102, DOPE, DMG-PEG2K, and CHOL. The formulations are shown in Table 5, and the preparation and characterization methods are 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 the following day for imaging. Data were analyzed using JMP Pro 16.0 software, employing standard least squares and effect screening for model fitting. The analysis results are shown below. Figure 3 (liver) and Figure 4 (spleen).

[0105] Table 5. Physicochemical characterization results of nanoparticles prepared with different formulations

[0106]

[0107] From Table 5 and Figure 3 , Figure 4 It is evident that considering SM-102 in the range of 40%–50% and DMG-PEG2K in the range of 1.5%–2.2%, there is no significant effect on transfection distribution; considering DOPE in the range of 5%–16%, with the increase of usage, its distribution in the spleen gradually increases and its enrichment in the liver gradually decreases, showing a significant trend.

[0108] Example 6: Evaluation of Immune Cell Targeting Effect

[0109] Delivery vectors with different lipid ratios were synthesized using GFP mRNA according to the method in Example 1.

[0110] 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. The mice were dissected 24 hours later, and spleen cells were collected, isolated, 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. Figure 5 .

[0111] Table 6. Physicochemical characterization results of nanoparticles prepared with different formulations

[0112]

[0113] 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.

[0114] Example 7: Evaluation of Immunological Effect

[0115] 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. Figure 6 As shown.

[0116] Table 7. Physicochemical characterization results of nanoparticles prepared with different formulations

[0117]

[0118] 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 nucleic acid drugs, comprising the following lipid components based on 100% of the total molar amount of lipids in the composition: Ionizable lipids: ≥40%, ≤50%; PEG-modified lipids: ≥0.5% and ≤3.0%; Steroids: ≥30%, ≤44.5%; Cofactor phospholipids: greater than 15% and less than 22%.

2. The composition according to claim 1, wherein the composition comprises the following lipid components, based on the total molar amount of lipids in the composition being 100%: Ionizable lipids: ≥40%, ≤50%; PEG-modified lipids: ≥1.0% and ≤2.5%; Steroids: ≥30%, ≤40%; Co-phospholipids: 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 an ionizable amino group; More preferably, the ionizable lipid is a compound with the structure shown in Formula I or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof: in: 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; R2 and R3 are independently substituted or unsubstituted C1-C 18 Straight-chain alkylene; 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; 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.

4. The composition according to claim 3, wherein, In Formula I: L1 and L2 are connecting bonds or divalent connecting groups, and each of the divalent connecting groups 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-; R2 and R3 are independently substituted or unsubstituted C3-C9 straight-chain alkylene groups; 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; R1 is H, -R 11 or -R 11 -OH, where R 11 It is a C1-C6 straight-chain alkyl group.

5. The composition according to claim 3, wherein, 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-yl8-((2-hydroxyethyl)(5-((2-methy l-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)hexyl2-hexyldecanoate, Heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecylthio)hexyl)amino)octanoate, or one or more of their tautomers and stereoisomers.

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

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

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

9. The use of the composition according to any one of claims 1-8 in the preparation of nucleic acid drug nanocarriers.

10. A nucleic acid drug nanocarrier, which is prepared by loading a nucleic acid drug onto the composition according to any one of claims 1-9.

11. The nucleic acid drug nanocarrier according to claim 10, wherein the particle size is 50nm-200nm, preferably 60nm-160nm; the encapsulation efficiency is greater than 80%; the potential is -15mV to +15mV, preferably -10mV to +10mV; and the pKa is 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 50,000 nucleotides, or 200 to 20,000 nucleotides, or 300 to 10,000 nucleotides, or 500 to 9,000 nucleotides.

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

14. A method for preparing a nucleic acid drug nanocarrier, the method comprising: An ethanol solution containing the lipid composition of any one of claims 1-8 is prepared and mixed with an aqueous buffer containing a nucleic acid drug to prepare a nanocarrier loaded with a nucleic acid drug.

15. The use of the nucleic acid drug nanocarrier according to any one of claims 10-13 or the nucleic acid drug nanocarrier prepared by the method of claim 14 in the preparation of nucleic acid drugs.

16. The application according to claim 15, wherein, The nucleic acid drug is used to target and induce protein expression in subjects.

17. The application according to claim 15, wherein: The nucleic acid drug is in the form of an intravenous injection or intramuscular injection, and is used to deliver the nucleic acid drug into the immune cells of a subject; or The nucleic acid drug is in the form of an intramuscular injection, and is intended for delivery to a non-liver site (e.g., spleen) in the subject; or The nucleic acid drug is an intra-articular injection formulation, used to deliver the nucleic acid drug to the joint site of the subject; or The nucleic acid drug is a tumor tissue injection formulation, which is used to deliver the nucleic acid drug to the tumor site of the subject; preferably, the tumor is a solid tumor, such as a subcutaneous tumor.

18. The application according to any one of claims 15-17, wherein, The nucleic acid drug is not liver-targeting.

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

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