CN118059061A - Preparation method and application of lipid nanoparticle for efficiently delivering nucleic acid drug - Google Patents

Abstract

The invention relates to a lipid nanoparticle for efficiently delivering nucleic acid drugs, and a preparation method and application thereof, and belongs to the technical field of medicines. The lipid nanoparticle of the present invention comprises a carrier and an encapsulated nucleic acid; the carrier comprises complex ionizable lipid, auxiliary phospholipid, cholesterol substance and polyethylene glycol lipid; the nucleic acid comprises one or more of mRNA, circular RNA, siRNA, microRNA, antisense nucleic acid and plasmid. Complex ionizable lipids include ionizable lipids with high efficiency in cell entry and ionizable lipids with long alkyl chains and high efficiency in membrane fusion. The compound ionizable lipid can obviously improve the mRNA expression efficiency of the commercialized four-component LNP through synergistic effect. The lipid nanoparticle can efficiently express nucleic acid drugs entrapped in the lipid nanoparticle after intramuscular injection administration, can efficiently activate humoral and cellular immune responses, and has important application prospects in the fields of herpes zoster and other vaccines.

Description

A preparation method and application of lipid nanoparticles for efficient delivery of nucleic acid drugs

Technical Field

The technical field of the present invention is the field of medical technology, and specifically relates to a five-component lipid nanoparticle (LNP) that can efficiently deliver nucleic acid drugs, and a preparation method and application thereof.

Background technique

In just two years, more than 3 billion doses of COVID-19 mRNA vaccines have been administered worldwide. Compared with traditional protein, virus, and deoxyribonucleic acid (DNA) vaccines, mRNA vaccines have the advantages of high safety, short R&D cycle, high production efficiency, encoding multiple proteins, and no need for adjuvants. However, due to its large molecular weight and negative charge, mRNA itself is difficult to enter cells, and mRNA itself has poor stability and is easily degraded by nucleases in the body. Therefore, a suitable delivery vector is needed to assist mRNA in entering target cells to work.

Lipid nanoparticles (LNP) are the only clinically approved mRNA vaccine carriers, which are composed of ionizable lipids, auxiliary phospholipids, cholesterol and pegylated lipids. LNP can effectively deliver mRNA into cells after intramuscular injection, activate cellular immunity and humoral immune response, and produce effective immune protection. The delivery efficiency of LNP is the key to the efficacy of mRNA vaccines, and the low lysosomal escape efficiency of LNP after entering the cell is the key to limiting the delivery efficiency of LNP. Therefore, it is urgent to develop new carriers that can increase the lysosomal escape efficiency of LNP and improve the delivery efficiency of LNP.

Summary of the invention

The present invention provides a lipid nanoparticle (hereinafter referred to as LNP), comprising a carrier and an encapsulated nucleic acid, wherein the carrier comprises a composite ionizable lipid, an auxiliary phospholipid, a cholesterol-like substance and a pegylated lipid; the nucleic acid comprises but is not limited to one or more of mRNA, circular RNA, siRNA, microRNA, antisense nucleic acid and a plasmid.

According to an embodiment of the present invention, the complex ionizable lipid accounts for 20 mol% to 70 mol% of the total lipids in the LNP, such as 30 mol%, 40 mol%, 50 mol%, 60 mol%.

According to an embodiment of the present invention, the complex ionizable lipid comprises a first ionizable lipid and a second ionizable lipid.

According to an embodiment of the present invention, the molar ratio of the first ionizable lipid to the second ionizable lipid is 1:99 to 99:1, preferably 2:8 to 8:2, such as 3:7, 4:6, 5:5, 6:4, 7:3, and more preferably 6:4.

According to an embodiment of the present invention, the first ionizable lipid and the second ionizable lipid are different and are independently selected from 8-[(2-hydroxyethyl)(6-oxo-6-decyloxyhexyl)amino]octanoic acid (heptadecan-9-yl) ester (SM-102), [(4-hydroxybutyl)azepinediyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), 4(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester (DLin MC3DMA), 3,6-bis{4-[bis(2-hydroxydodecyl)amino]butyl}piperazine-2,5-dione (cKK-E12), 9-(4-(dimethylamino)butyryloxy)heptadecanedioic acid di((Z)-non-2-en-1-yl) ester (L319), N2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), 8-[(2-hydroxyethyl)(8-nonyloxy-8-oxooctyl)amino]octanoic acid (heptadecan-9-yl) ester (Lipid5), 1,1'-[(2-{4-[2-({2-[bis(2-hydroxydodecyl)amino]ethyl}(2-hydroxydodecyl)amino)ethyl One or more of [(methyl] piperazin-1-yl} ethyl) azadialkyl] bis(dodecane-2-ol) (C12-200), (2,3 dioleoylpropyl) trimethylammonium chloride (DOTAP), dimethyldioctadecyl ammonium bromide (DDAB), tetrakis(8-methylnonyl) 3',3',3",3"-{[(methyl azadialkyl) bis(propane-3,1-diyl)] bis(azatriyl)} tetrapropionate (306Oi10); preferably 8[(2-hydroxyethyl)(6-oxo-6-decyloxyhexyl) amino] caprylic acid (heptadecanyl) ester (SM-102) or [(4-hydroxybutyl) azadiyl] bis(hexane 6,1-diyl) bis(2-hexyldecanoate) (ALC-0315).

In some embodiments, the complex ionizable lipid includes 3,6-bis{4-[bis(2-hydroxydodecyl)amino]butyl}piperazine-2,5-dione (cKK-E12) and 9-(4-(dimethylamino)butyryloxy)heptadecanedioic acid di((Z)-non-2-en-1-yl) ester (L319), and the molar ratio thereof is 1:99 to 99:1, preferably 2:8 to 8:2, for example 3:7, 4:6, 5:5, 6:4, 7:3.

According to an embodiment of the present invention, the auxiliary phospholipid accounts for 2 mol% to 20 mol% of the total lipids in the LNP, for example 4 mol%, 8 mol%, 10 mol%, 12 mol%, 4 mol%, 16 mol%, 18 mol%.

According to an embodiment of the present invention, the auxiliary phospholipid includes but is not limited to 1,2-distearoyl-sn-glyceryl-3-phosphatidylcholine (DSPC), 1,2-dioleoyl-sn-glyceryl-3-phosphatidylcholine (DOPC), 1,2-dipalmitoyl-sn-glyceryl-3-phosphatidylcholine (DPPC), 2-oleoyl-1-palmitoyl-sn-glyceryl-3-phosphatidylcholine (POPC), 1,2-dioleoyl-sn-glyceryl-3-phosphatidylethanolamine (DOPE), 2-oleoyl-1-palmitoyl-sn-glyceryl-3-phosphatidylethanolamine (POPE), 1,2-distearoyl-sn-glyceryl-3-phosphatidylethanolamine (DSPE), 1,2-dipalmitoyl-sn-glyceryl-3-phosphatidylethanolamine (DPPE), preferably DSPC.

According to an embodiment of the present invention, the cholesterol-like substance accounts for 10 mol% to 60 mol% of the total lipids in the LNP, for example, 20 mol%, 30 mol%, 40 mol%, 50 mol%.

According to an embodiment of the present invention, the cholesterol-like substance is selected from cholesterol and its derivatives.

According to an embodiment of the present invention, the cholesterol and its derivatives include but are not limited to one or more of cholesterol, β-sitosterol, cholestanol, cholestanone, cholestenone, 7β-hydroxycholesterol, 7α-hydroxycholesterol, preferably cholesterol.

According to an embodiment of the present invention, the PEGylated lipid accounts for 0.3 mol% to 30 mol% of the total lipid in the LNP, preferably 0.5 mol% to 2.5 mol%, such as 1.0 mol%, 1.5 mol%, 2.0 mol%.

According to an embodiment of the present invention, the PEGylated lipids include but are not limited to one or more of 1,2-dimyristoyl-rac-glyceryl-3-methoxypolyethylene glycol (DMG-PEG), 1,2-distearoyl-rac-glyceryl-3-methoxypolyethylene glycol (DSG-PEG), 1,2-dipalmitoyl-rac-glyceryl-3-methoxypolyethylene glycol (DPG-PEG), and 1,2-distearoyl-sn-glyceryl-3-phosphatidylethanolamine-methoxypolyethylene glycol (DSPE-PEG), preferably DMG-PEG.

In the present invention, the above-mentioned "total lipids" refers to the sum of complex ionizable lipids, auxiliary phospholipids, cholesterol substances and PEGylated lipids.

According to an embodiment of the present invention, the nucleic acid selected by the present invention includes at least one of CpG-FAM, mFLuc (mRNA encoding firefly luciferase), mOVA (mRNA encoding chicken ovalbumin), and mVZV (mRNA encoding herpes zoster gE protein).

According to an embodiment of the present invention, the encapsulation rate of the nucleic acid is 30% to 99%, preferably 70%-90%, such as 70%, 75%, 80%, 85%, 90%.

According to an embodiment of the present invention, the molar ratio of the nitrogen element contained in the complex ionizable lipid to the phosphorus element contained in the nucleic acid is (1-50):1; preferably (5-10):1, and exemplarily 5.67:1.

According to an embodiment of the present invention, the hydrated particle size of the lipid nanoparticles is less than 200 nm, such as 80 to 180 nm.

According to an embodiment of the present invention, the polydispersity coefficient of the lipid nanoparticles is less than 0.2, for example, 0.119, 0.102, or 0.093.

In some embodiments, the molar ratio of the complex ionizable lipid: helper phospholipid: cholesterol-like substance: polyethylene glycol lipid is 50:10:38.5:1.5.

In some embodiments, the complex ionizable lipid includes a first ionizable lipid and a second ionizable lipid in a ratio of 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, or 2:8.

The nucleic acid is CpG-FAM, mFLuc, mOVA or mVZV.

The present invention also provides a method for preparing the lipid nanoparticles, which comprises preparing the lipid nanoparticles by blending an aqueous phase containing nucleic acids and a lipid organic phase through a blending method or a microfluidics method.

According to an embodiment of the present invention, the lipid organic phase is prepared as follows: the composite ionizable lipid, auxiliary phospholipid, cholesterol-like substance and pegylated lipid are dissolved in an organic solvent in proportion to obtain an organic phase. Preferably, the organic solvent is an organic solvent that can dissolve the above substances, such as anhydrous ethanol.

According to an embodiment of the present invention, the preparation method of the aqueous phase containing nucleic acid is as follows: dissolving the nucleic acid in a buffer solution to obtain an aqueous phase. Preferably, the buffer solution can be a buffer solution with a pH of 4 to 6, for example, a sodium citrate buffer solution with a pH of 4 or 5.5.

According to an embodiment of the present invention, the blending method includes the following steps: using a pipette to absorb the aqueous phase containing nucleic acids, quickly adding it to the lipid organic phase, and then quickly blowing the mixed liquid for more than 30 times, blowing it evenly and letting it stand at room temperature for 10 minutes, and dialyzing the resulting product after standing, for example, dialyzing it in a PBS buffer solution with a volume greater than 1000 times for more than 6 hours to obtain the lipid nanoparticles.

According to an embodiment of the present invention, the microfluidic method includes the following steps: fixing syringes containing an aqueous phase and a lipid organic phase on a syringe pump respectively, injecting the aqueous phase: lipid organic phase into a microfluidic chip at a volume ratio of 3:1 and mixing thoroughly, collecting the mixed solution after mixing and standing at room temperature for 10 minutes, and dialyzing the resulting product with a PBS buffer solution after standing, for example, dialyzing in a PBS buffer solution with a volume greater than 1000 times for more than 6 hours to obtain the lipid nanoparticles.

The present invention also provides the use of the lipid nanoparticles in the preparation of biological preparations.

According to an embodiment of the present invention, the biologic is an injectable biologic.

In some embodiments, the biologic is a vaccine, preferably an mRNA vaccine.

According to an embodiment of the present invention, the administration method of the biological preparation is intramuscular injection.

According to an embodiment of the present invention, the biological agent is used to prevent herpes zoster.

The present invention also provides a biological agent having the meaning as described above.

The beneficial effects of the present invention are as follows:

The LNP prepared by the present invention has uniform particle size, good dispersibility, high encapsulation rate, good biocompatibility, low toxicity and side effects, and can efficiently deliver nucleic acid drugs. The preparation method of the LNP of the present invention has simple operation steps and is easy to mass produce.

The present invention combines a first ionizable lipid and a second ionizable lipid to form a composite ionizable lipid, which has an ionizable lipid with high efficiency in entering cells and an ionizable lipid with a long alkyl chain and high efficiency in membrane fusion ability. The composite ionizable lipid of the present invention can significantly improve the mRNA expression efficiency of the commercial four-component LNP through synergistic effect.

The present invention uses composite ionizable lipids to prepare five-component LNPs, which have a higher activation efficiency on antigen-presenting cells in draining lymph nodes and spleen than commercial four-component LNPs after intramuscular injection.

The five-component LNP prepared by the composite ionizable lipids of the present invention can efficiently deliver the herpes zoster mRNA vaccine. After intramuscular injection, it significantly activates the cellular immunity and humoral immune response in the body, produces a large number of antigen-specific binding antibodies in the blood, and increases the antigen-specific T cell content in the spleen, which has an excellent protective effect. It is expected to achieve a breakthrough in the field of herpes zoster mRNA vaccines and has great application prospects.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 : Chemical structures of the first ionizable lipid and the second ionizable lipid used in the examples of the present invention.

FIG. 2 : Hydration particle size distribution curve of LNP obtained in Example 1B of the present invention.

FIG3 : Cryo-TEM image of the five-component LNP obtained in Example 1A of the present invention.

FIG. 4 shows the bioluminescence intensity of DC2.4 cells transfected with LNPs loaded with mFluc in Example 3 of the present invention.

FIG5 : The membrane fusion ability of the five-component LNP was detected using mouse red blood cells in Example 4 of the present invention.

Figure 6: In vivo imaging of small animals was used to verify the bioluminescence intensity of the five-component LNP loaded with mFLuc at the injection site of mice in Example 5 of the present invention. (a) In vivo imaging image and statistical value results of bioluminescence intensity at the injection site 4 hours after intramuscular injection; (b) In vivo imaging image and statistical value results of bioluminescence intensity at the injection site 24 hours after intramuscular injection.

Figure 7: Flow chart of animal experiment in Example 6 of the present invention.

FIG8 : Activation efficiency of different LNPs loaded with mOVA on DC cells in draining lymph nodes and spleen in Example 6 of the present invention.

Figure 9: Flow chart of animal experiment in Example 7 of the present invention.

FIG. 10A shows the antibody titers of antigen-specific IgG in the serum of mice at days 14, 28 and 35 after the first intramuscular injection of LNPs loaded with mVZV in Example 7 of the present invention.

10B : Antigen-specific IgG1 antibody titers in the serum of mice on days 14, 28 and 35 after the first intramuscular injection of LNPs loaded with mVZV in Example 7 of the present invention.

10C : Antigen-specific IgG2c antibody titers in the serum of mice on days 14, 28 and 35 after the first intramuscular injection of LNPs loaded with mVZV in Example 7 of the present invention.

Figure 11: The number of antigen-specific splenocytes secreting IFN-γ in the spleen of mice on the 42nd day after the first intramuscular injection of LNPs loaded with mVZV in Example 7 of the present invention (a) ELISpot spot number statistics; (b) optical photograph of the ELISpot well.

Figure 12: The number of antigen-specific CD4 ⁺ T cells secreting IFN-γ in the spleen of mice on day 42 after the first intramuscular injection of LNPs loaded with mVZV in Example 7 of the present invention.

Figure 13: Concentrations of IL-2 and IFN-γ secreted by antigen-specific spleen cells on day 42 after the first intramuscular injection of LNPs encapsulating mVZV in mice in Example 7 of the present invention.

Figure 14: Body weight monitoring curve of mice during immunization in Example 8 of the present invention.

FIG. 15 : H&E staining of tissue and organ sections on day 42 after the first intramuscular injection of LNPs loaded with mVZV in mice in Example 8 of the present invention.

Detailed ways

The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

In the LNP prepared in the following example, the molar ratio of nitrogen element in the composite ionizable lipid to phosphorus element in the encapsulated nucleic acid drug is 5.67:1, and the volume ratio of the aqueous phase to the organic phase is 3:1; wherein the aqueous phase refers to the nucleic acid drug dissolved in a 50mM sodium citrate buffer solution at pH = 4, wherein the concentration of the nucleic acid drug is 0.17mg/mL; the organic phase refers to the composite ionizable lipid: auxiliary phospholipid: cholesterol substance: PEGylated lipid dissolved in anhydrous ethanol at a molar ratio of 50:38.5:10:1.5, wherein the molar ratio of the first ionizable lipid to the second ionizable lipid is 1:99 to 99:1, preferably 2:8 to 8:2, for example, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, and more preferably 6:4.

The composite ionizable lipid includes a first ionizable lipid and a second ionizable lipid, which are 3,6-bis{4-[bis(2-hydroxydodecyl)amino]butyl}piperazine-2,5-dione, abbreviated as cKK-E12; 9-(4-(dimethylamino)butyryloxy)heptadecanedioic acid di((Z)-non-2-en-1-yl) ester, abbreviated as L319; the auxiliary phospholipid is 1,2-distearoyl-sn-glyceryl-3-phosphocholine, abbreviated as DSPC; and the polyethylene glycol lipid is 1,2-dimyristoyl-sn-glyceryl-3-methoxypolyethylene glycol 2000, abbreviated as DMG-PEG2000. The structural formulas of the first ionizable lipid and the second ionizable lipid are shown in Figure 1.

Example 1A

Five-component lipid nanoparticles (referred to as five-component LNPs) were prepared as follows:

(1) dissolving a nucleic acid drug with FAM fluorescently modified CPG DNA (FAM-CPG) in a sodium citrate buffer solution at the above concentration to form an aqueous phase;

(2) The composite ionizable lipid, auxiliary phospholipid, cholesterol and pegylated lipid are dissolved in anhydrous ethanol in the above proportions to form an organic phase, wherein the molar ratio of cKK-E12 to L319 is 6:4; the aqueous phase and the organic phase are mixed by a blending method or a microfluidics method, and after the mixing is completed, the obtained product is dialyzed using a PBS buffer solution to prepare lipid nanoparticles.

Example 1B

The preparation method of the five-component lipid nanoparticles of this embodiment is basically the same as that of Example 1A, except that the nucleic acid drug is mRNA encoding firefly luciferase (mFLuc), and the molar ratio of cKK-E12 and L319 is 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, and 8:2.

Example 1C

The preparation method of the five-component lipid nanoparticles of this embodiment is basically the same as that of Example 1A, except that the nucleic acid drug is mRNA encoding chicken ovalbumin (mOVA), and the molar ratio of cKK-E12 and L319 is 6:4.

Example 1D

The preparation method of the five-component lipid nanoparticles of this embodiment is basically the same as that of Example 1A, except that the nucleic acid drug is mRNA encoding the herpes zoster virus gE protein (mVZV), and the molar ratio of cKK-E12 and L319 is 6:4.

Comparative Examples 1 to 3

The preparation methods of the lipid nanoparticles of Comparative Examples 1 to 3 are basically the same as those of Example 1B, except that:

In Comparative Example 1, the composite ionizable lipid was replaced with MC3, i.e., a four-component LNP with MC3 as the ionizable lipid;

In Comparative Example 2, the composite ionizable lipid was replaced with cKK-E12, i.e., a four-component LNP with cKK-E12 as the ionizable lipid;

In Comparative Example 3, the composite ionizable lipid was replaced with L319, i.e., a four-component LNP with L319 as the ionizable lipid.

Example 2: Characterization of Hydrated Particle Size of Lipid Nanoparticles

Take 100 μL of the five-component lipid nanoparticles prepared in Examples 1A to 1D, and use dynamic light scattering to determine the hydrated particle size of the lipid nanoparticles in PBS buffer solution, where Figure 2 shows the five-component LNP of Example 1B and the four-component LNP of Comparative Example 1 (denoted as MC3); the hydrated particle size and polydispersity coefficient of the five-component LNP of Example 1A are 140 nm and 0.09, respectively, the hydrated particle size and polydispersity coefficient of the five-component LNP of Example 1C are 110 nm and 0.1, respectively, and the hydrated particle size and polydispersity coefficient of the five-component LNP of Example 1D are 130 nm and 0.1, respectively.

10 μL of the five-component lipid nanoparticles prepared in Example 1 (the molar ratio of cKK-E12 and L319 was 6:4) was taken, and the morphology of the lipid nanoparticles was photographed using a cryo-transmission electron microscope, wherein FIG3 is the LNP of Example 1A.

The hydrated particle sizes of the five-component lipid nanoparticles prepared in Examples 1A to 1D are all less than 200 nm, and the polydispersity coefficient is less than 0.2, indicating that the preparation method of the present invention can form stable lipid nanoparticles with uniform particle size.

Example 3: Screening of five-component LNPs using in vitro transfection

The five-component LNPs loaded with mFLuc prepared in Example 1B were used, and the four-component LNPs of Comparative Examples 1 to 3 were selected as a control group to screen different ratios of cKK-E12 and L319 as ionizable lipids to act synergistically.

The experimental steps are as follows: DC2.4 cells were incubated overnight in a 96-well plate at 20,000 cells per well. When the cell confluence reached 50%, LNPs of Example 1B and Comparative Examples 1 to 3 were added respectively, and cultured in a saturated humidity incubator at 37°C and 5% _CO2 concentration for 24 hours. An Untreated cells group (referring to cells without any treatment after plating) and a FreemRNA group (referring to mRNA without a carrier being added directly to the cell culture medium) were also set up. The culture medium was removed, cell lysate and firefly luciferase substrate were added, and after shaking for 10 minutes, the bioluminescence value was read using an ELISA instrument. The test results are shown in Figure 4.

From this verification, it can be seen that the five-component LNP with the synergistic effect of cKK-E12 and L319 in a molar ratio of 6/4 has the highest mRNA expression efficiency.

Example 4: Verification of the membrane fusion ability of the five-component LNP

The mFLuc-encapsulated five-component LNP prepared in Example 1 was used, and the four-component LNP of Comparative Examples 1 to 3 was selected as a control group to detect the membrane fusion ability of the five-component LNP.

The experimental steps are as follows: mouse red blood cells are placed in a transparent 96-well plate, sodium citrate buffer solution of pH 5.5 and PBS buffer solution of pH 7.4 are added respectively, and then LNPs of Example 1 and Comparative Examples 1 to 3 are added correspondingly and incubated at 37°C for 1 hour, and a negative control group (i.e., only buffer solution and red blood cells are added to the well plate, recorded as Untreated cells) and a positive control group (i.e., buffer solution, red blood cells and 0.5% Triton X-100 are added to the well plate, recorded as 0.5% Triton X-100) are incubated at 37°C for 1 hour. After the incubation is completed, the supernatant is centrifuged and the absorbance value of the supernatant at 540nm is measured. The degree of hemolysis of red blood cells is judged by the absorbance value. The test results are shown in Figure 5.

This demonstrates that the membrane fusion ability of the five-component LNP of the present invention is between that of the LNP containing only cKK-E12 or the LNP containing only L319.

Example 5: Verification of mRNA expression efficiency of five-component LNP in vivo

The mFLuc-encapsulated five-component LNP prepared in Example 1 was used, and the four-component LNP of Comparative Examples 1 to 3 were selected as a control group to detect the mRNA expression efficiency of the five-component LNP of the present invention in vivo.

The experimental steps are as follows: female C57BL/6 mice aged 6-8 weeks were selected, and 2 μg of LNP of Example 1A and Comparative Examples 1 to 3 were administered to each mouse in each group by intramuscular injection. 200 μL of 200 mg/mL firefly luciferase substrate was injected intraperitoneally into each mouse 4 hours and 24 hours after administration, respectively. After 15 minutes, the bioluminescence intensity in the mice was detected using small animal in vivo imaging.

The above experiments verified that after using the five-component LNP of Example 1, the injection site had the strongest bioluminescence intensity at both 4 hours and 24 hours, see Figure 6, where (a) is the bioluminescence intensity at 4 hours, and (b) is the bioluminescence intensity at 24 hours.

It can be seen from this that the five-component LNP of the present invention has a high mRNA expression efficiency in vivo.

Example 6: Verification of the DC cell activation efficiency of the five-component LNP in vivo

The mOVA-encapsulated five-component LNP prepared in Example 1C was used, and the four-component LNP of Comparative Example 1 was selected as a control group to detect the DC cell activation efficiency of the five-component LNP of the present invention in vivo.

The experimental steps are as follows: 8-week-old female C57BL/6 mice were selected, with 4 mice in each experimental group. Each group of mice was immunized by intramuscular injection on day 0 and day 7, and each mouse in each group was administered 10 μg of LNP of Example 1C and Comparative Example 1, respectively. A PBS group was also set up in which only PBS buffer solution was injected; on day 8, the mice were killed, and the draining lymph nodes and spleen at the injection site were removed in vitro. For the experimental process, see Figure 7. After the lymph node cells and spleen cells were processed separately, a single cell suspension was obtained, and the number of activated DC cells (CD11c ⁺ CD80 ⁺ , CD11c ⁺ CD86 ⁺ ) in the spleen and draining lymph nodes was detected by flow cytometry, see Figure 8.

This demonstrates that the mRNA-loaded five-component LNP of the present invention has a high DC cell activation efficiency in the draining lymph nodes and spleen of mice.

Example 7: Verification of the immune efficacy of five-component LNP in vivo

The five-component LNP encapsulating mVZV prepared in Example 1D was used, wherein the four-component LNP of Comparative Example 1, the inactivated VZV virus vaccine (hereinafter referred to as the inactivated vaccine) and the herpes zoster recombinant gE protein subunit vaccine plus AS01 adjuvant (hereinafter referred to as the subunit vaccine) were selected as the control group of this experiment to detect the immune efficacy of the five-component LNP of the present invention in vivo.

The experimental steps are as follows: 8-week-old female C57BL/6 mice were selected, with 4 mice in each experimental group, and each group of mice was immunized by intramuscular injection at week 0 and week 3, and each mouse in each group was administered 10 μg of LNP, inactivated vaccine and subunit vaccine of Example 1D and Comparative Example 1, respectively, and a PBS group was set up in which only PBS buffer solution was injected; the mice were bled from the eye sockets at weeks 2, 4 and 5, and the serum was separated, and the mice were killed at week 6. The experimental process is shown in Figure 9. The antibody titers of herpes zoster-specific IgG, IgG1 and IgG2c in the serum of mice on days 14, 28 and 35 were measured by ELISA, and the test results are shown in Figures 10A to 10C.

After the mouse spleen was processed, a single cell suspension was obtained, and 300,000 cells were placed in each well of the ELISpot plate and stimulated with the herpes zoster gE peptide library for 24 hours, and then the number of spleen cells that specifically secreted IFN-γ was detected by color development. The test results are shown in Figure 11, where (a) ELISpot spot number statistics, (b) ELISpot well optical photo. Splenocytes were placed in a 96-well plate with 1 million cells per well, stimulated with the herpes zoster gE peptide library for 5 hours, and protein transport inhibitors were added. The number of herpes zoster-specific T cells (CD3 ⁺ CD4 ⁺ CD45 ⁺ IFN-γ ⁺ ) was detected by flow cytometry, and the test results are shown in Figure 12. Splenocytes were then placed in a 96-well plate with 1 million cells per well, stimulated with the herpes zoster gE peptide library for 48 hours, centrifuged to obtain the cell supernatant, and the amount of IL-2 and IFN-γ secreted by spleen cells in the cell supernatant was measured by ELISA, and the test results are shown in Figure 13.

This demonstrates that the mRNA-loaded five-component LNP of the present invention has the ability to efficiently activate specific cellular immunity and humoral immunity in vivo.

Example 8: Verification of the toxicity of five-component LNP in vivo

The five-component LNPs encapsulating mVZV prepared in Example 1D were used, and the four-component LNPs, inactivated vaccine and subunit vaccine of Comparative Example 1 were selected as the control group of this experiment to detect the vaccine efficacy of the five-component LNPs in vivo.

The experimental steps are as follows: 8-week-old female C57BL/6 mice were selected, with 4 mice in each experimental group. Each group of mice was immunized by intramuscular injection at week 0 and week 3. Each mouse in each group was administered 10 μg of the five-component LNP in Example 1D and the four-component LNP, inactivated vaccine and subunit vaccine in Comparative Example 1, respectively. A PBS group was set up in which only PBS buffer solution was injected. The weight of the mice was monitored during the immunization period. See Figure 14 for the experimental process. The mice were killed in week 6, and the heart, liver, spleen, lung, kidney, brain and injection site muscle of the mice were embedded in paraffin for H&E staining sections. See Figure 15 for the test results.

This demonstrates that the five-component LNP containing mRNA of the present invention has low toxicity in vivo.

The above is a description of the exemplary embodiments of the present invention. However, the protection scope of the present application is not limited to the above embodiments. Any modification, equivalent substitution, improvement, etc. made by those skilled in the art within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims (10)

1. A lipid nanoparticle comprising a carrier and an encapsulated nucleic acid, the carrier comprising a complex ionizable lipid, a helper phospholipid, a cholesterol species, and a pegylated lipid; the nucleic acids include one or more of mRNA, circular RNA, siRNA, microRNA, antisense nucleic acids, and plasmids.

2. The lipid nanoparticle of claim 1, wherein the complex ionizable lipid comprises 20mol% to 70mol% of the total lipids in the LNP.

Preferably, the complex ionizable lipid comprises a first ionizable lipid and a second ionizable lipid.

Preferably, the molar ratio of the first ionizable lipid to the second ionizable lipid is from 1:99 to 99:1.

And/or, the first and second ionizable lipids being different from each other and being selected from the group consisting of (heptadec-9-yl) 8- [ (2-hydroxyethyl) (6-oxo-6-decyloxyhexyl) amino ] octanoate, [ (4-hydroxybutyl) azadiyl ] bis (hexane-6, 1-diyl) bis (2-hexyldecanoate), 4 (N, N-dimethylamino) butanoic acid (diimine) methyl ester, 3, 6-bis {4- [ bis (2-hydroxydodecyl) amino ] butyl } piperazine-2, 5-dione, 9- (4- (dimethylamino) butanoyloxy) heptadecanedioic acid di ((Z) -non-2-en-1-yl) ester, N2, 2-diiminoethyl- [1,3] -dioxolane, 8- [ (2-hydroxyethyl) (8-nonyloxy-8-oxooctyl) amino ] octanoate, 1' - [ (2-hydroxydodecyl) amino ] piperazine-2, 5-dione, 9- (4- (dimethylamino) butanoyl) heptadecanedioic acid di ((Z) -non-2-en-1-yl) ethyl) 2, 2-diiminoethyl- [ 1-2- ({ -hydroxyethyl ] amino ] octanoate, one or more of (2, 3 dioleoyl propyl) trimethyl ammonium chloride, dimethyl dioctadecyl ammonium bromide, tetra (8-methylnonyl) 3',3', 3"- { [ (methylazadialkyl) bis (propane-3, 1 diyl) ] bis (azatriyl) ] tetrapropionate.

3. The lipid nanoparticle according to claim 1 or 2, wherein the helper phospholipid comprises 2 to 20mol% of the total lipid in the LNP.

Preferably, the auxiliary phospholipids comprise one or more of 1, 2-distearoyl-sn-glycero-3-phosphatidylcholine, 1, 2-dioleoyl-sn-glycero-3-phosphatidylcholine, 1, 2-dipalmitoyl-sn-glycero-3-phosphatidylcholine, 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphatidylcholine, 1, 2-dioleoyl-sn-glycero-3-phosphatidylethanolamine, 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphatidylethanolamine, 1, 2-distearoyl-sn-glycero-3-phosphatidylethanolamine, 1, 2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine.

4. A lipid nanoparticle according to any one of claims 1 to 3, wherein the cholesterol species comprises from 10mol% to 60mol% of the total lipid in the LNP.

Preferably, the cholesterol-based substance is selected from cholesterol and derivatives thereof.

Preferably, the cholesterol and derivatives thereof comprise one or more of cholesterol, beta-sitosterol, cholestanol, cholestanone, cholestenone, 7beta-hydroxycholesterol, 7alpha-hydroxycholesterol.

5. The lipid nanoparticle of any one of claims 1-4, wherein the pegylated lipid comprises between 0.3mol% and 30mol% of the total lipids in the LNP.

Preferably, the pegylated lipid comprises one or more of 1, 2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol, 1, 2-distearoyl-rac-glycero-3-methoxypolyethylene glycol, 1, 2-dipalmitoyl-rac-glycero-3-methoxypolyethylene glycol, 1, 2-distearoyl-sn-glycero-3-phosphatidylethanolamine-methoxypolyethylene glycol.

The "total lipid" refers to the sum of complex ionizable lipids, helper phospholipids, cholesterol species, and pegylated lipids.

6. The lipid nanoparticle of any one of claims 1-5, wherein the nucleic acid has an encapsulation efficiency of 30-99%.

Preferably, the molar ratio of the nitrogen element contained in the complex ionizable lipid to the phosphorus element contained in the nucleic acid is (1 to 50): 1.

Preferably, the lipid nanoparticle has a hydrated particle size of less than 200nm.

Preferably, the lipid nanoparticle has a polydispersity of less than 0.2.

7. The method for preparing the lipid nanoparticle according to any one of claims 1 to 6, wherein the method comprises preparing the lipid nanoparticle by a blending method or a microfluidic method of an aqueous phase containing nucleic acid and a lipid organic phase.

Preferably, the preparation method of the lipid organic phase is as follows: the complex ionizable lipid, the auxiliary phospholipid, the cholesterol substance and the polyethylene glycol lipid are dissolved in an organic solvent according to a proportion to obtain an organic phase.

Preferably, the preparation method of the aqueous phase containing nucleic acid comprises the following steps: the nucleic acid is dissolved in a buffer solution to obtain an aqueous phase. Preferably, the buffer solution can be selected from buffer solutions with pH of 4-6.

8. The method of claim 7, wherein the blending process comprises the steps of: and sucking the water phase containing the nucleic acid by using a pipetting gun, rapidly adding the water phase into the lipid organic phase, then rapidly blowing the mixed liquid for more than 30 times, standing at room temperature for 10 minutes after uniform blowing, and dialyzing the obtained product after standing to obtain the lipid nanoparticle.

Preferably, the microfluidic method comprises the steps of: the syringes containing the aqueous and lipid organic phases were separately mounted on syringe pumps with the aqueous phase: and injecting the lipid organic phase into the microfluidic chip according to the volume ratio of 3:1, fully mixing, collecting the mixed solution after mixing, standing for 10 minutes at room temperature, and dialyzing the obtained product by using PBS buffer solution after standing to obtain the lipid nanoparticle.

9. Use of the lipid nanoparticle of any one of claims 1-6 in the preparation of a biological agent.

Preferably, the biological agent is an injectable biological agent.

Preferably, the biologic is administered by intramuscular injection.

Preferably, the biological agent is for the prevention of shingles.

10. A biologic which is the biologic of claim 9.