LIPID COMPOUND AND USE THEREOF IN DELIVERY OF NUCLEIC ACID

Abstract

Provided are an ionizable lipid compound with an adjacent cis-double bond structure, a preparation method therefor, and the use thereof in the delivery of an active therapeutic agent (e.g., a nucleic acid). The ionizable lipid compound can provide a higher encapsulation rate of active substances and a better cell or in vivo transfection rate, and is particularly suitable for preparing nanoparticles with a solid structure.

Claims

1. A compound having a structure ##STR00183## of formula I, wherein: Q is a substituted or unsubstituted linear C2-20 alkylene, wherein one or more C atoms of the alkylene are optionally substituted by heteroatom(s) independently selected from O, S, and N; or Q is a substituted or unsubstituted saturated or unsaturated 4- to 6-membered ring, wherein ring atoms of the 4- to 6-membered ring optionally comprise one or more heteroatoms independently selected from O, S, and N; a substituent group for the substitution is selected from halogen, OH, linear or branched C1-20 alkyl, linear or branched C1-20 alkoxy, linear or branched C2-20 alkenyl, linear or branched C2-20 alkynyl, CH.sub.2CH(OH)R.sub.5, and ##STR00184## R.sub.1, R.sub.2, R.sub.3, and R.sub.4 can be identical or different and are each independently selected from hydrogen, substituted or unsubstituted linear or branched C1-30 alkyl, substituted or unsubstituted linear or branched C2-30 alkenyl, substituted or unsubstituted linear or branched C2-30 alkynyl, and CH.sub.2CH(OH)R.sub.5, wherein one or more C atoms of the alkyl, alkenyl, or alkynyl are optionally substituted by heteroatom(s) independently selected from O, S, and N; a substituent group for the substitution is selected from halogen, OH, linear or branched C1-10 alkyl, and linear or branched C1-10 alkoxy; provided that at least one of R.sub.1, R.sub.2, R.sub.3, and R.sub.4 is ##STR00185## R.sub.5 is selected from hydrogen, substituted or unsubstituted linear or branched C1-30 alkyl, substituted or unsubstituted linear or branched C2-30 alkenyl, and substituted or unsubstituted linear or branched C2-30 alkynyl, wherein one or more C atoms of the alkyl, alkenyl, or alkynyl are optionally substituted by heteroatom(s) independently selected from O, S, and N; a substituent group for the substitution is selected from halogen, OH, linear or branched C1-10 alkyl, and linear or branched C1-10 alkoxy; R.sub.6 is selected from hydrogen, C1-3 alkyl, C1-3 alkoxy, and OH; n is selected from integers from 1 to 8, m is selected from integers from 0 to 8, and n and m are independent of each other and can be identical or different; when at least two of R.sub.1, R.sub.2, R.sub.3, and R.sub.4 are ##STR00186## n and m in each of the groups are independent of each other and can be identical or different.

2. The compound according to claim 1, characterized in that, Q is ##STR00187## wherein R.sub.8 and R.sub.9 are each independently selected from substituted or unsubstituted linear C1-10 alkylene, wherein one or more C atoms of the alkylene are optionally substituted by heteroatom(s) independently selected from O, S, and N; R.sub.7 is hydrogen, halogen, OH, linear or branched C1-20 alkyl, linear or branched C2-20 alkenyl, linear or branched C2-20 alkynyl, CH.sub.2CH(OH)R.sub.5, or ##STR00188## a substituent group for the substitution is halogen, OH, linear or branched C1-10 alkyl, or linear or branched C1-10 alkoxy.

3. The compound according to claim 1, characterized in that, Q is ##STR00189## wherein x and y are identical or different and are independently selected from integers from 1 to 8; preferably, x and y are identical or different and are selected from integers from 1 to 3; preferably, R.sub.7 is linear or branched C1-4 alkyl.

4. The compound according to claim 1, characterized in that, R.sub.6 is OH.

5. The compound according to claim 1, characterized in that, n is selected from integers from 4 to 8, and m is selected from integers from 4 to 8.

6. The compound according to claim 1, characterized in that, the compound is selected from compounds of the following formulas A, B, C, and D: ##STR00190## wherein each n.sub.1 is independent and is identical or different, and each n.sub.1 is selected from integers from 1 to 8; each m.sub.1 is independent and is identical or different, and each m.sub.1 is selected from integers from 0 to 8; preferably, each n.sub.1 is selected from integers from 4 to 8, and each m.sub.1 is selected from integers from 4 to 8; preferably, each n.sub.1 is identical, and each m.sub.1 is identical; ##STR00191## wherein each n.sub.2 is independent and is identical or different, and each n.sub.2 is selected from integers from 1 to 8; each m.sub.2 is independent and is identical or different, and each m.sub.2 is selected from integers from 0 to 8; preferably, each n.sub.2 is selected from integers from 4 to 8, and each m.sub.2 is selected from integers from 4 to 8; preferably, each n.sub.2 is identical, and each m.sub.2 is identical; ##STR00192## wherein each n.sub.3 is independent and is identical or different, and each n.sub.3 is selected from integers from 1 to 8; each m.sub.3 is independent and is identical or different, and each m.sub.3 is selected from integers from 0 to 8; preferably, each n.sub.3 is selected from integers from 4 to 8, and each m.sub.3 is selected from integers from 4 to 8; preferably, each n.sub.3 is identical, and each m.sub.3 is identical; and ##STR00193## wherein each n.sub.4 is independent and is identical or different, and each n.sub.4 is selected from integers from 1 to 8; each m.sub.4 is independent and is identical or different, and each m.sub.4 is selected from integers from 0 to 8; preferably, each n.sub.4 is selected from integers from 4 to 8, and each m.sub.4 is selected from integers from 4 to 8; preferably, each n.sub.4 is identical, and each m.sub.4 is identical; preferably, ##STR00194##

7. Use of the compound according to claim 1 in preparing a bioactive substance delivery system, wherein preferably, the delivery system is microparticles, nanoparticles, liposomes, lipid nanoparticles, or microbubbles.

8. A bioactive substance delivery system comprising the compound according to claim 1, wherein preferably, the delivery system is microparticles, nanoparticles, liposomes, lipid nanoparticles, or microbubbles.

9. A pharmaceutical composition comprising the bioactive substance delivery system according to claim 8.

10. A preparation method for the compound according to claim 1, wherein the method is as follows: ##STR00195## comprising 1) in the presence of a reductant, reducing the carboxyl group of compound Al to a hydroxyl group to give compound A2; 2) oxidation: in the presence of an oxidant, oxidizing the hydroxyl group of compound A2 to an aldehyde group to give compound A3; 3) halogenation-reduction: firstly subjecting an aldehyde a-hydrogen of compound A3 to a halogenation reaction with a halogenating reagent under an acidic condition to give an a-halogenated aldehyde intermediate, and then in the presence of a reductant, reducing the aldehyde group of the ?-halogenated aldehyde to a hydroxyl group to give compound A4; 4) epoxidation: in the presence of a base, subjecting compound A4 to an intramolecular nucleophilic substitution reaction to give epoxide A5; and 5) ring-opening reaction: subjecting compound A5 to a ring-opening reaction with an amine to give a final compound.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0043] FIG. 1 shows the pKa curve of ionizable lipid II-37;

[0044] FIG. 2 shows the cell transfection efficiency of mRNA-encapsulated LNP formed by ionizable lipid II-37;

[0045] FIG. 3 shows the cell transfection efficiency of pDNA-encapsulated LNP formed by ionizable lipid II-37;

[0046] FIG. 4 shows the cell transfection efficiency of siRNA-encapsulated LNP formed by ionizable lipid II-37;

[0047] FIG. 5 shows the comparison of cell transfection efficiencies of mRNA-encapsulated LNPs formed by ionizable lipid II-37 and commercial molecule MC3, respectively;

[0048] FIG. 6 shows that the loaded mRNA is labeled with Cy5, the mRNA-encapsulated LNPs are formed by ionizable lipid II-37 and commercial molecule MC3, respectively, and the efficiencies of the LNPs in delivering mRNA into cells are observed by fluorescence staining;

[0049] FIG. 7 shows the comparison of cell transfection efficiencies of mRNA-encapsulated LNPs formed by ionizable lipid II-37 and C14-113, respectively; and

[0050] FIG. 8 shows the cytotoxicity of II-37-LNP and C14-113-LNP determined by MTT method.

DETAILED DESCRIPTION

[0051] The technical solutions of the present invention will be further illustrated in detail with reference to the following specific examples. It should be understood that the following examples are merely exemplary illustrations and explanations of the present invention, and should not be construed as limiting the protection scope of the present invention. All techniques implemented based on the content of the present invention described above are included within the protection scope of the present invention.

[0052] Unless otherwise stated, the raw materials and reagents used in the following examples are all commercially available products or can be prepared using known methods.

Example 1: Synthesis of Ionizable Lipid II-37

[0053] ##STR00181##

[0054] Synthesis of linoleyl alcohol (a2): LiAlH.sub.4 (7.20 g) and linoleic acid (50 g, al) were added to tetrahydrofuran (950 mL) at 0? C., and then the mixture was stirred at 25? C.for 2 h. After the reaction was completed, as detected by thin layer chromatography (TLC), water (7.2 mL), an aqueous NaOH solution (7.2 mL, mass fraction: 15%), and water (21.6 mL) were sequentially added to the reaction solution to quench the reaction, and an appropriate amount of Na.sub.2SO.sub.4 was added. After being stirred for 15 min, the mixture was filtered through a Buchner funnel, the filter cake was washed with ethyl acetate, and the filtrate was collected and concentrated by evaporation to give the target product linoleyl alcohol (a2, 47.4 g).

[0055] .sup.1H NMR (400 MHZ, CDCl.sub.3): ? 5.27-5.44 (m, 4 H), 3.63 (t, J=6.63 Hz, 2 H), 2.77 (t, J=6.44 Hz, 2 H), 1.97-2.12 (m, 4 H), 1.57-1.63 (m, 1 H), 1.20-1.46 (m, 18 H), 0.83-0.95 (m, 3 H)

[0056] Synthesis of (9Z,12Z)-octadeca-9,12-dienal (a3): Linoleyl alcohol (25.0 g, a2) and 2-iodoxybenzoic acid (39.4 g) were added to acetonitrile (170 mL) at room temperature, and then the mixture was stirred at 85? C. for 4 h. The reaction solution was filtered through a Buchner funnel, the filter cake was washed with dichloromethane, and the filtrate was collected and concentrated by evaporation to give the target product (9Z,12Z)-octadeca-9,12-dienal (a3, 24.0 g).

[0057] .sup.1H NMR (400 MHZ, CDCl.sub.3): ? 9.76 (t, J=1.76 Hz, 1 H), 5.25-5.43 (m, 4 H), 2.76 (t, J=6.17 Hz, 2 H), 2.41 (td, J=7.33, 1.87 Hz, 2 H), 2.04 (q, J=6.84 Hz, 4 H), 1.56-1.68 (m, 2 H), 1.22-1.36 (m, 14 H), 0.88 (t, J=6.73 Hz, 3 H)

[0058] Synthesis of (9Z,12Z)-2-chloro-octadeca-9,12-dien-1-ol (a4): (9Z,12Z)-Octadeca-9,12-dienal (43.0 g, a3), DL-proline (5.62 g), and N-chlorosuccinimide were added to acetonitrile (246 mL) at 0? C., and then the mixture was stirred at 0? C. for 2 h. After the reaction was completed, the reaction solution was diluted with absolute ethanol (246 mL), sodium borohydride (8.8 g) was added, and then the mixture was stirred at 0? C. for 4 h. The reaction mixture was quenched with water (120 mL) and extracted with methyl tert-butyl ether. The organic phases were combined, washed with saturated brine, dried over sodium sulfate, filtered, and concentrated by evaporation to give the target product (9Z,12Z)-2-chloro-octadeca-9,12-dien-1-ol (a4, 46 g), which was used directly in the next step.

[0059] .sup.1H NMR (400 MHZ, CDCl.sub.3): ? 5.25-5.51 (m, 4 H), 3.97-4.07 (m, 1 H), 3.79 (dd, J=12.01, 3.63 Hz, 1 H), 3.59-3.70 (m, 1 H), 2.67-2.90 (m, 2 H), 1.96-2.15 (m, 5 H), 1.64-1.82 (m, 1 H), 1.20-1.49 (m, 15 H), 0.89 (br t, J=6.75 Hz, 3 H)

[0060] Synthesis of 2-[(7Z,10Z)-hexadeca-7,10-dien-1-yl]oxirane (a5): (9Z,12Z)-2-Chloro-octadeca-9,12-dien-1-ol (45 g, a4) and an aqueous sodium hydroxide solution (120 g sodium hydroxide was dissolved in 585 mL of water) were added to 1,4-dioxane (450 mL) at room temperature. After the dropwise addition, the mixture was stirred at 35? C. for 2 h. After the reaction was completed, as detected by TLC, the reaction solution was separated by a separating funnel, washed with saturated brine, dried over sodium sulfate, filtered, and concentrated by evaporation, and then the residue was purified by flash column chromatography using petroleum ether/ethyl acetate as an eluent to give the target product 2-[(7Z,10Z)-hexadeca-7,10-dien-1-yl]oxirane (a5, 29.11 g).

[0061] .sup.1H NMR (400 MHZ, CDCl.sub.3): ? 5.27-5.46 (m, 4 H), 2.87-2.98 (m, 1 H), 2.70-2.85 (m, 3 H), 2.46 (dd, J=5.00, 2.75 Hz, 1 H), 1.94-2.21 (m, 4 H), 1.24 -1.58 (m, 17 H), 0.78-1.00 (m, 3 H)

[0062] Synthesis of II-37: 2-[(7Z,10Z)-hexadeca-7,10-dien-1-yl]oxirane (5 g) and N,N-bis(2-aminoethyl)methylamine (739 mg) were added to ethanol (10 mL) at room temperature, and the mixture was stirred at 90? C. for 36 h. The reaction solution was concentrated by evaporation, and the residue was purified by flash column chromatography using dichloromethane/methanol as an eluent to give crude product II-37 (4 g). The target product was again purified by flash column chromatography with dichloromethane/methanol to give II-37 (2.2 g).

[0063] .sup.1H NMR (400 MHz, CDCl.sub.3): ? 5.27-5.44 (m, 12 H), 3.48-3.79 (m, 3 H), 2.63-3.00 (m, 12 H), 2.16-2.61 (m, 12 H), 2.05 (q, J=6.80 Hz, 12 H), 1.18-1.57 (m, 51 H), 0.89 (t, J=6.88 Hz, 9 H)

[0064] ESI-MS: m/z 910.8 [M+H].sup.+, 911.8 [M+2H].sup.+, 912.8 [M+3H].sup.+

Example 2: Dissociation Constant (pKa) of Ionizable Lipid II-37

[0065] Ionizable lipids have two main roles: binding to nucleic acids and allowing the release of the nucleic acid molecules in cells. The pKa of lipids is an important factor, because the lipids need to be positively charged at a low pH value to bind to nucleic acids, but not charged at a neutral pH value, such that the formed LNPs do not cause toxicity. The ionizable lipid II-37 was determined to have a pKa of 6.81 by a TNS dye-binding assay. The results are shown in FIG. 1.

Example 3: Preparation of Lipid Nanoparticles by II-37 Encapsulating mRNA

[0066] Ionizable lipid II-37, DSPC, CHOL, and DMG-PEG2000 were dissolved in ethanol according to a molar ratio of 35%:15%:48.5%:1.5% as an organic phase, and Lucferase mRNA (LucRNA) was dissolved in an aqueous solution with the pH of 4 as an aqueous phase. A nanoparticle suspension was prepared by microfluidic technology on a nanomedicine manufacturing instrument (PrecisionNanoSystems Inc. (PNI), Canada, model: Ignite) according to a volume ratio of aqueous phase to organic phase of 3:1. After the preparation was completed, an ultrafiltration concentration was performed on the suspension to give the final LucRNA-LNP lipid nanoparticle, which was stored at 2? C. to 8? C. for later use.

[0067] The particle size and Zeta potential of LucRNA-LNP were characterized by a Zetasizer Pro nanoparticle size potentiometer (Malvern Panalytical). The encapsulation efficiency of LucRNA-LNP was detected by the Ribogreen method using an F-280 fluorescence spectrophotometer (Tianjin Gangdong Sci.&Tech. Co. Ltd). The CHO cells transfection efficiency of the prepared LucRNA-LNP was detected by a fluorescein reporter gene assay using a multi-mode microplate reader (BioTek, model: SLXFATS). The method for in vitro transcription of LucRNA was as follows: CHO-KI cells were plated at a cell density of 2.5?10.sup.5 cells/mL, and transfection was performed when the cell confluence was 30%-50%. 2 ?g of LucRNA was added to each well for transfection, and the positive control was transfected using a transfection reagent Lipofectamine MessagerMAX (ThermoFisher Scientific). The transfection operation was performed according to a product instruction of the transfection reagent. After 48 h of transfection, the protein expression level was detected by the multi-mode microplate reader. The negative control was a cell culture medium without LucRNA-LNP. The detection results in Example 3 are shown in Table 2.

TABLE-US-00002 TABLE 2 Particle size Zeta potential Encapsulation (nm) PDI (mV) efficiency (%) RLU (2 ?g/mL) LucRNA-LNP 108.66 0.13 17.87 96.4% 1088112

[0068] It can be seen from the results in Example 3 that the particle size of the lipid nanoparticle LucRNA-LNP prepared by the combination of the novel lipid compound is about 108 nm, the particle size distribution of LucRNA-LNP is relatively narrow (PDI is relatively small), and the encapsulation efficiency is up to 96%. The in vitro cell transfection efficiency is up to 1 million. The results show that the mRNA-encapsulated LNP prepared from the ionizable lipid II-37 not only has very good physicochemical parameters, but also has extremely high cell transfection efficiency.

[0069] Further, the HEK293T cells transfection efficiency of LucRNA-LNP prepared by the same LNP was detected by the fluorescein reporter gene assay using the multi-mode microplate reader (BioTek, model: SLXFATS), and the amount of transfected LucRNA was 0.5 ?g, 1.0 ?g, and 2.0 ?g, respectively. The method for in vitro transcription of LucRNA was as follows: HEK293T cells were plated at a cell density of 2.5?10.sup.5 cells/mL, and transfection was performed when the cell confluence was 30%-50%. The positive control was transfected with 0.5 ?g of LucRNA using a transfection reagent Lipofectamine 2000 (ThermoFisher Scientific). The transfection operation was performed according to a product instruction of the transfection reagent. After 48 h of transfection, the protein expression level was detected by the multi-mode microplate reader. The negative control was a cell culture medium without LucRNA-LNP. The in vitro cell transfection efficiency is shown in FIG. 2, indicating that mRNA-encapsulated LNP prepared from the ionizable lipid II-37 has extremely high cell transfection efficiency, which is about 10 times higher than that of the commercial Lipofectamine 2000 when the same amount of mRNA was transfected.

Example 4: Preparation of Lipid Nanoparticles by II-37 Encapsulating DNA

[0070] Ionizable lipid II-37, DSPC, CHOL, and DMG-PEG2000 were dissolved in ethanol according to a molar ratio of 45%: 10%:43.5%:1.5% as an organic phase, and Lucferase DNA (pDNA) was dissolved in an aqueous solution with the pH of 4 as an aqueous phase. A nanoparticle suspension was prepared by microfluidic technology on a nanomedicine manufacturing instrument (PNI, Canada, model: Ignite) according to a volume ratio of aqueous phase to organic phase of 3:1. After the preparation was completed, an ultrafiltration concentration was performed on the suspension to give the final pDNA-LNP lipid nanoparticle, which was stored at 2? C. to 8? C.for later use.

[0071] The particle size and Zeta potential of pDNA-LNP were characterized by a Zetasizer Pro nanoparticle size potentiometer (Malvern Panalytical). The detection results in Example 4 are shown in Table 3, showing that the particle size of the lipid nanoparticle pDNA-LNP prepared by the combination of the novel lipid compound is about 173 nm, and the particle size distribution of pDNA-LNP is relatively narrow (PDI is relatively small).

TABLE-US-00003 TABLE 3 Particle size (nm) PDI Zeta potential (mV) pDNA-LNP 173.2 0.213 24.1

[0072] The 293T cells transfection efficiency of the prepared pDNA-LNP was detected by a fluorescein reporter gene assay using a multi-mode microplate reader (BioTek, model: SLXFATS), and the amount of transfected pDNA was 0.5 ?g, 1.0 ?g, and 2.0 ?g, respectively. The method for in vitro transcription was as follows: 293T cells were plated at a cell density of 2.0?10.sup.5 cells/mL, and transfection was performed when the cell confluence was 30%-50%. The positive control was transfected with 2 ?g of pDNA using a transfection reagent Lipofectamine 2000 (ThermoFisher Scientific). The transfection operation was performed according to a product instruction of the transfection reagent. After 48 h of transfection, the protein expression level was detected by the multi-mode microplate reader. The negative control was a cell culture medium without pDNA-LNP. The in vitro cell transfection efficiency is shown in FIG. 3, indicating that DNA-encapsulated LNP prepared from the ionizable lipid II-37 has extremely high cell transfection efficiency: the protein expression level of 1.0 ?g of pDNA transfected with the LNP prepared from II-37 is higher than that of 2 ?g of pDNA transfected with Lipofectamine 2000; the in vitro cell transfection efficiency of II-37 is about 3 times higher than that of the commercial Lipofectamine 2000 when the same 2 ?g of pDNA was transfected.

Example 5: Preparation of Lipid Nanoparticles by II-37 Encapsulating siRNA

[0073] Ionizable lipid II-37, DSPC, CHOL, and DMG-PEG2000 were dissolved in ethanol according to a molar ratio of 45%:15%:38.5%:1.5% as an organic phase, and Lucferase siRNA (siRNA) was dissolved in an aqueous solution with the pH of 4 as an aqueous phase. A nanoparticle suspension was prepared by microfluidic technology on a nanomedicine manufacturing instrument (PNI, Canada, model: Ignite) according to a volume ratio of aqueous phase to organic phase of 3:1. After the preparation was completed, an ultrafiltration concentration was performed on the suspension to give the final siRNA-LNP lipid nanoparticle, which was stored at 2? C. to 8? C. for later use.

[0074] The particle size and Zeta potential of siRNA-LNP were characterized by a Zetasizer Pro nanoparticle size potentiometer (Malvern Panalytical). The detection results in Example 5 are shown in Table 4. The particle size of the lipid nanoparticle siRNA-LNP prepared by the combination of the novel lipid compound is about 294 nm.

TABLE-US-00004 TABLE 4 Particle size (nm) PDI Zeta potential (mV) siRNA-LNP 294.0 0.318 20.3

[0075] The 293T cells transfection efficiency of the prepared siRNA-LNP was detected by a fluorescein reporter gene assay using a multi-mode microplate reader (BioTek, model: SLXFATS), and the amount of transfected siRNA was 0.5 ?g, 1.0 ?g, and 2.0 ?g, respectively. The method for in vitro transcription was as follows: Lucferase reporter stably transfected 293T cells were plated at a cell density of 2.0?10.sup.5 cells/mL, and transfection was performed when the cell confluence was 30%-50%. The positive control was transfected with 1.0 ?g of siRNA using a transfection reagent Lipofectamine 2000 (ThermoFisher Scientific). The transfection operation was performed according to a product instruction of the transfection reagent. After 24 h of transfection, the protein expression level was detected by the multi-mode microplate reader. The negative control was a cell culture medium without siRNA-LNP. The in vitro cell transfection efficiency is shown in FIG. 4, indicating that siRNA-encapsulated LNP prepared from the ionizable lipid II-37 has extremely high protein knockdown efficiency.

Example 6: Effect Comparison of II-37 and Commercial Ionizable Cationic Lipid Molecule MC3

[0076] The molecular formula of MC3 was (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(N,N-dimethylamino)butanoate.

[0077] Lipid nanoparticles were prepared according to the method described in Example 3 using II-37 and MC3, respectively, at the following specific molar ratios: II-37:DSPC:CHOL:DMG-PEG2000=45:15:38.5:1.5; MC3:DSPC:CHOL:DMG-PEG2000=45:15:38.5:1.5; the N/P ratio was 5:1.

[0078] The physicochemical and quality control data of the prepared lipid nanoparticles are shown in the following table:

TABLE-US-00005 Sample information Particle size (nm) PDI Zeta potential Encapsulation efficiency mRNA-LNP (II-37) 154.58 0.1068 22.07 90.5 mRNA-LNP (MC3) 234.08 0.1259 2.44 40.7

[0079] It can be seen from the above table that the encapsulation efficiency of the lipid nanoparticle prepared from II-37 is up to 90.5%, which is much higher than that of the lipid nanoparticle prepared from MC3, and the lipid nanoparticle has smaller and more uniform particle size and higher potential.

[0080] When the prepared lipid nanoparticles were transfected into CHO-K1 cells according to the same transfection method as in Example 3, the expression of protein was known. The results are shown in FIG. 5. Under the same amount of transfected mRNA, after the lipid nanoparticle prepared from II-37 (shown as C2 in the figure) carrying mRNA was transfected into the cells, the expression of protein in the cells was much higher than that of MC3, indicating that the lipid nanoparticle prepared from II-37 has very high cell transfection efficiency.

[0081] In addition, Cy5-mRNA-LNP (II-37) and Cy5-mRNA-LNP (MC3) were obtained by labeling the loaded mRNA with Cy5. After 2 h and 6 h of incubation with 293T cells, cellular lysosomes were stained with LysoSensor? Green, and the effect of Cy5-mRNA entering into the cells was observed.

[0082] It can be seen from FIG. 6 that after Cy5-mRNA-LNP (II-37) was incubated with the cells for 6 h, most of Cy5-mRNA reached lysosomes with a colocalization coefficient of 0.626; after Cy5-mRNA-LNP (MC3) was incubated with the cells for 6 h, Cy5-mRNA was less likely to enter into the cells. It can be seen from the comparison that the nucleic acid delivery efficiency of the II-37 molecule is better than that of the MC3 molecule.

[0083] It can be seen from the results in Example 6 that the lipid nanoparticles prepared by the combination of the novel lipid compound are superior to the MC3 molecule in both nucleic acid delivery efficiency and in vitro cell transfection efficiency.

Example 7: Comparison of II-37 and its Structural Analog Molecule C14-113

[0084] The structural formula of C14-113 was as follows:

##STR00182##

[0085] Lipid nanoparticles were prepared according to the method described in Example 3 using II-37 and C14-113, respectively, at the following specific molar ratios: II-37:DSPC:CHOL:DMG-PEG2000=45:15:38.5:1.5; C14-113:DSPC:CHOL:DMG-PEG2000=45:15:38.5:1.5; the N/P ratio was 10:1.

[0086] The physicochemical and quality control data of the prepared lipid nanoparticles are shown in the following table:

TABLE-US-00006 Sample information Particle size (nm) PDI Zeta potential mRNA-LNP (II-37-LNP) 136.68 0.14 20.07 mRNA-LNP (C14-113-LNP) 152.65 0.12 24.1

[0087] When the prepared lipid nanoparticles were transfected into 293T cells according to the same transfection method as in Example 3, the expression of protein was known. The results are shown in FIG. 7. Under the same amount of transfected mRNA, after the lipid nanoparticle prepared from II-37 (shown as II-37-LNP in the figure) carrying mRNA was transfected into the cells, the expression of protein in the cells was much higher than that of C14-113, indicating that the lipid nanoparticle prepared from II-37 has very high cell transfection efficiency.

[0088] In addition, the cytotoxicity of II-37-LNP and C14-113-LNP was determined by MTT method, and the effect of factors such as vector dosage and action time on the proliferation of normal cells (293T cells) was examined. The results are shown in FIG. 8. 48 h after the transfection of cells, the lipid nanoparticle prepared from II-37 (shown as II-37-LNP in the figure) carrying mRNA still maintained relatively good cell activity at a higher dose (2 ?g/mL), indicating that the cytotoxicity of the lipid nanoparticle prepared from II-37 is very low.

[0089] It can be seen from the results in Example 7 that the lipid nanoparticles prepared by the combination of the novel lipid compound have low cytotoxicity and are superior to the structural analog molecule C14-113 in mRNA transfection efficiency.

[0090] The embodiments of the present invention have been described above. However, the present invention is not limited to the embodiments described above. Any modification, equivalent, improvement, and the like made without departing from the spirit and principle of the present invention shall fall within the protection scope of the present invention.