Cationic lipid compound, preparation method and application thereof

Abstract

The present invention discloses a cationic lipid compound, its preparation method, and its application. The synthesis of the cationic lipid compound in the present invention is unrestricted in the choice of isonitrile and can be performed normally without the presence of a catalyst. The method for synthesizing the cationic lipid compound in the present invention is simple, inexpensive, and easily applicable. Furthermore, the synthesized lipid molecular structures are diverse, and the synthesis yield is high, exceeding 70%. Gene vectors prepared using the cationic lipid compound in the present invention exhibit good gene transfection efficiency and low cytotoxicity, and have potential for clinical application.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical chemistry, and in particular relates to a cationic lipid compound and a preparation method and application thereof. Background Art

[0002] Gene therapy has shown tremendous potential in the diagnosis and treatment of a variety of diseases, including viral infections, vaccines, neurological disorders, inflammation, and cancer. In gene therapy, because DNA is easily degraded by nucleases in the body and cannot directly enter cells through contact with cell membranes, a vector is required to bind to the exogenous DNA and transport and protect it during its transport within the body. Currently, gene vectors primarily include viral and non-viral vectors. Viruses can integrate their genomes into target cells, and while viral vectors typically deliver genetic material with high transfection efficiency, they suffer from drawbacks such as high immunogenicity, high toxicity, limited targeting, restrictions on DNA size and target cell types, and high cost, limiting their clinical application. Compared to viral vectors, non-viral gene vectors offer advantages such as low toxicity, ease of preparation, chemical modification, and mass production, and have garnered significant attention in recent years. Non-viral vectors primarily include lipid nanoparticles (LNPs), cationic polymers, inorganic nanomaterials, and natural polysaccharide carriers.

[0003] LNPs are vesicles composed of a mixture of cationic lipids and helper lipids (such as DOPE, DOPC, cholesterol, and polyethylene glycol esters) in a specific ratio. The positive charge on the surface of the cationic lipids binds to the negatively charged gene through electrostatic interactions, forming a stable complex that protects the gene. Helper lipids enhance gene delivery efficiency by improving LNP biocompatibility, structural stability, and serum stability.

[0004] The structure of cationic lipids is an important factor affecting the gene transport performance of LNPs. First, the positive charge of the lipid molecule is obtained by protonation of the nitrogen atom of the amino group on the hydrophilic head. Different types and numbers of amino groups will result in different positive charges, resulting in different transfection activities and cytotoxicity. The type, length and unsaturation of the hydrophobic tail chain will affect the lipophilicity, biocompatibility, transition temperature, pKa, etc. of the LNP / DNA complex, which in turn affects the transfection efficiency of the material. The linking bonds of lipid molecules usually have good physiological stability and biodegradability under specific conditions, that is, they maintain structural stability during circulation in the body and rapidly degrade after reaching the target site, effectively releasing the gene while reducing cytotoxicity. The most common linking groups include ester bonds, amide bonds, carbamate bonds, phosphates, ether bonds, etc.

[0005] Currently, there are many methods for synthesizing cationic lipids, but most of them still have problems such as complex synthesis routes, low synthesis yields, high costs, and difficulty in large-scale production.

[0006] A method for simultaneously coupling primary / secondary amines, ketones, and isonitriles via the Ugi three-component reaction has been reported to synthesize lipid molecules in one step. The lipid structure consists of an amine head group, an isonitrile linker, and an alkyl or alkylene ketone lipid tail chain. Compared with traditional lipids synthesized using multi-step reactions, this method has the advantages of simple and rapid synthesis. When α-acidic isonitriles are used as reactants, isonitriles can act as both electrophiles and nucleophiles. The reaction does not require a catalyst and can be carried out in a mixed solvent of dichloromethane and ethanol. However, the synthesis yield is relatively low, reaching only 34% at most. For other isonitriles, the reaction requires a Lewis acid (such as phenylphosphite, AlCl3, SbCl5, etc.) as a catalyst. Summary of the Invention

[0007] In response to the above-mentioned existing technologies, the present invention provides a cationic lipid compound and its preparation method and application to solve technical problems such as poor transport performance of existing gene carriers, low synthesis yield of lipid compounds for preparing gene carriers, and the need for Lewis acid as a catalyst for the reaction.

[0008] In order to achieve the above object, the technical solution adopted by the present invention is to provide a cationic lipid compound, the structural formula of the cationic lipid compound is shown in Formula I,

[0009]

[0010] Wherein, R1 is a nitrogen heterocyclic group or an amino group, R2 is a substituted phenyl group or a straight-chain alkyl group; and R3 is a straight-chain hydrocarbon group.

[0011] On the basis of the above technical solution, the present invention can also be improved as follows.

[0012] Furthermore, the nitrogen heterocyclic group is piperidinyl or pyrrolidinyl, the amino group is N-methylethylamino; the substituted phenyl group is nitro-substituted phenyl; the straight-chain alkyl group is n-tridecyl; and the straight-chain hydrocarbon group is n-dodecyl or oleyl.

[0013] Furthermore, the cationic lipid compound is one of the following compounds:

[0014]

[0015] The present invention also discloses a method for preparing the cationic lipid compound, which comprises the following steps:

[0016] S1: dissolving the acid, aldehyde and isonitrile in a first organic solvent, reacting at 40-50°C for 16-32 hours, then removing the solvent and purifying to obtain product A;

[0017] The acid is one of the following:

[0018]

[0019] The aldehyde is one of the following:

[0020]

[0021] Isonitrile is one of the following isonitriles:

[0022]

[0023] S2: dissolving product A in a second organic solvent, adding trifluoroacetic acid in an ice bath, then heating to room temperature and keeping the temperature to react for 5 to 8 hours; then spin-drying the solvent and removing the trifluoroacetic acid to obtain the product.

[0024] The preparation method can be further improved on the basis of the above technical solution.

[0025] Furthermore, the molar ratio of the acid, the aldehyde and the isonitrile is 1:1:1.

[0026] Furthermore, the first organic solvent is tetrahydrofuran; and the second organic solvent is dichloromethane.

[0027] Furthermore, the purification method in S1 is column chromatography, and the eluent used in the column chromatography is obtained by mixing petroleum ether and ethyl acetate in a volume ratio of 3:1.

[0028] Furthermore, the method for removing trifluoroacetic acid in S2 is: dissolving the substance after the solvent is spin-dried in a second organic solvent, and then spin-dried; repeating the above operation 4 to 6 times.

[0029] The present invention also discloses the use of a cationic lipid compound in the preparation of a gene vector. The vector is prepared by the following steps: adding the cationic lipid compound and DOPE in a molar ratio of 1:1-2 to a reaction vessel, dissolving them in anhydrous chloroform, then spinning off the solvent to obtain a thin film adhered to the side wall of the reaction vessel, and vacuum drying overnight; then adding ultrapure water to the reaction vessel, heating at 70°C for 30 minutes to remove the thin film, and then pulverizing in an ice bath to obtain a gene vector solution.

[0030] The beneficial effects of the present invention are:

[0031] 1. The gene vector prepared by the cationic lipid compound of the present invention has good gene transfection efficiency and low cytotoxicity, and is a gene vector with excellent performance.

[0032] 2. The method for synthesizing cationic lipid compounds in the present invention is simple, inexpensive, and easy to promote and apply.

[0033] 3. There are no restrictions on the selection of isonitriles when synthesizing cationic lipid compounds in the present invention, and the synthesis can proceed normally without the participation of a catalyst. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The reaction equation for preparing the cationic lipid compound of the present invention is:

[0035] Figure 2 is the particle size and Zeta potential distribution diagram of LNP;

[0036] Figure 3 The gel electrophoresis results of LNP;

[0037] Figure 4 The results of cytotoxicity test of LNP (B1, B2) and pGL-3 plasmid complex in HepG2 cells;

[0038] Figure 5 The results of cytotoxicity test of LNP (B3, B4, B5) and pGL-3 plasmid complex in A549 cells;

[0039] Figure 6 The results of qualitative transfection experiments of LNP (B1, B2) and pEGFP-N1 plasmid complex in HepG2 cells with / without serum;

[0040] Figure 7 The results of qualitative transfection experiments of LNP (B3, B4, B5) and pEGFP-N1 plasmid complex in A549 cells with and without serum;

[0041] Figure 8 The results of quantitative transfection experiments of LNP (B1, B2) and pGL-3 plasmid complexes in HepG2 cells with and without serum;

[0042] Figure 9 These are the results of quantitative transfection experiments of LNP (B3, B4, B5) and pGL-3 plasmid complexes in A549 cells with and without serum. DETAILED DESCRIPTION

[0043] The reaction equation for preparing the cationic lipid compound in the present invention is as follows: Figure 1 The specific implementation of the present invention is described in detail below with reference to the embodiments.

[0044] Example 1

[0045] A cationic lipid compound, the structural formula of which is shown below:

[0046]

[0047] The cationic lipid compound in this embodiment was prepared by the following steps:

[0048] (1) 1-Boc-pyrrolidine-3-carboxylic acid (0.15 g, 0.7 mmol), o-nitrobenzaldehyde (0.11 g, 0.7 mmol) and oleyl isocyanide (0.19 g, 0.7 mmol) were dissolved in 2 mL of tetrahydrofuran, heated to 45°C and kept warm for 24 h. The solvent was then removed by distillation under reduced pressure, and the mixture was separated by column chromatography (eluent volume ratio: petroleum ether:ethyl acetate = 3:1) to obtain 0.53 g of a light yellow oily product A1, the structural formula of which is shown below, with a yield of 78.3%;

[0049]

[0050] (2) A1 (0.35 g, 0.55 mmol) was dissolved in 2 mL of dichloromethane, and trifluoroacetic acid (0.63 g, 5.5 mmol) was slowly added dropwise under ice bath conditions. After the addition was complete, the temperature was raised to room temperature and kept warm for 6 h. The reaction progress was monitored by plate counting. After the reaction was completed, the solvent was dried by vacuum distillation, and 3 mL of dichloromethane was added and continued to be dried. The above operation was repeated 5 times until all the trifluoroacetic acid was removed. The product B1 was obtained by vacuum drying as 0.34 g of a light yellow oil, i.e., the cationic lipid compound in this example, with a yield of 93%. The characterization results of B1 were as follows:

[0051] 1H-NMR: (400MHz, CDCl3): δ (ppm) 7.99 (d, 1H, Ar-H), 7.75 (m, 2H, Ar-H), 7.55 (t, 1H, Ar-H) ,6.59(d,1H,-COOCHCONH-),5.45-5.20(m,2H,-CH=CH-),3.42(s,2H,-NHCH2CH-),3.20(d, 2H,-CONHCH2-),2.32(m,4H,-CH2CH=),1.92(s,1H,-CH2CHCOO-),1.60(m,4H,-NHCH2CH2C H-),1.42(s,2H,-CONHCH2CH2-),1.23(d,24H,-CH2CH2-,-CH2CH3),0.85(t,3H,CH3CH2-).

[0052] HR-MS: [M+H] + :544.3751

[0053] Example 2

[0054] A cationic lipid compound, the structural formula of which is shown below:

[0055]

[0056] The cationic lipid compound in this embodiment was prepared by the following steps:

[0057] (1) 1-Boc-4-piperidinic acid (0.15 g, 0.65 mmol), o-nitrobenzaldehyde (0.10 g, 0.65 mmol) and oleyl isocyanide (0.18 g, 0.65 mmol) were dissolved in 2 mL of tetrahydrofuran, heated to 45°C and kept warm for 24 h. The solvent was then removed by distillation under reduced pressure, and the product A2 was separated by column chromatography (eluent volume ratio: petroleum ether:ethyl acetate = 3:1) to obtain 0.27 g of a light yellow oily product A2, the structural formula of which is shown below, with a yield of 76.5%.

[0058]

[0059] (2) A2 (0.27 g, 0.49 mmol) was dissolved in 2 mL of dichloromethane, and trifluoroacetic acid (0.56 g, 4.9 mmol) was slowly added dropwise under ice bath conditions. After the addition was complete, the temperature was raised to room temperature and kept warm for 6 h. The reaction progress was monitored by plate counting. After the reaction was completed, the solvent was evaporated by vacuum distillation, 3 mL of dichloromethane was added, and the reaction was continued to be evaporated. The above operation was repeated 5 times until all the trifluoroacetic acid was evaporated. The product B2 was obtained as a light yellow oily product (0.3 g), i.e., the cationic lipid compound in this example, with a yield of 90.2%. The characterization results of B2 were as follows:

[0060] 1H-NMR: (400MHz, CDCl3): δ (ppm) 8.00 (d, 1H, Ar-H), 7.77 (m, 2H, Ar-H), 7.56 (t, 1H, Ar-H ),6.64(d,1H,-COOCHCONH-),5.33(m,2H,-CH=CH-),3.54-3.04(m,6H,-NHCH2CH2-,-CON HCH2-),2.81(s,1H,-CH2CHCOO-),2.30-1.87(m,4H,-CH2CH=),1.60(s,4H,-NHCH2CH2CH -),1.42(t,2H,-CONHCH2CH2-),1.23(d,24H,-CH2CH2-,-CH2CH3),0.85(t,3H,CH3CH2-).

[0061] HR-MS: [M+H] + :558.3909

[0062] Example 3

[0063] A cationic lipid compound, the structural formula of which is shown below:

[0064]

[0065] The cationic lipid compound in this embodiment was prepared by the following steps:

[0066] (1) 1-Boc-pyrrolidine-3-carboxylic acid (0.15 g, 0.7 mmol), tetradecanal (0.15 g, 0.7 mmol) and dodecanonitrile (0.14 g, 0.7 mmol) were dissolved in 2 mL of tetrahydrofuran, heated to 45°C and kept warm for 24 h. The solvent was then removed by distillation under reduced pressure, and the mixture was separated by column chromatography (eluent volume ratio: petroleum ether:ethyl acetate = 3:1) to obtain 0.32 g of a white solid product A3, the structural formula of which is as follows, with a yield of 72%:

[0067]

[0068] (2) A3 (0.32 g, 0.5 mmol) was dissolved in 2 mL of dichloromethane, and trifluoroacetic acid (0.11 g, 5 mmol) was slowly added dropwise under ice bath conditions. After the addition was complete, the temperature was raised to room temperature and kept warm for 6 h. The reaction progress was monitored by plate counting. After the reaction was completed, the solvent was evaporated by vacuum distillation, 3 mL of dichloromethane was added, and the reaction was continued to be evaporated. The above operation was repeated 5 times until all the trifluoroacetic acid was evaporated. The product B3 was obtained by vacuum drying with a yield of 90.7%, which was a white solid product, i.e., the cationic lipid compound in this example. The characterization results of B1 were as follows:

[0069] 1 H-NMR: (400MHz, CDCl3): δ(ppm)5.02(m,1H,-COOCH-),3.62(m,2H,-NHCH2CH-),3.41(m,3H,-NHCH2CH2CH-,-NHCH2CH-),3.25(m, 2H,-COONHCH2-),2.38(m,2H,-NHCH2CH2CH-),1.81(m,2H,-COOCHCH2-),1.25(m,42H,-CH2CH2-,-CH2CH3),0.88(t,6H,CH3CH2-)

[0070] HR-MS: [M+H] + :523.4837

[0071] Example 4

[0072] A cationic lipid compound, the structural formula of which is shown below:

[0073]

[0074] The cationic lipid compound in this embodiment was prepared by the following steps:

[0075] (1) 1-Boc-4-piperidinic acid (0.15 g, 0.65 mmol), tetradecanal (0.14 g, 0.65 mmol), and dodecanonitrile (0.14 g, 0.65 mmol) were dissolved in 2 mL of tetrahydrofuran, heated to 45°C, and kept warm for 24 h. The solvent was then removed by distillation under reduced pressure, and the mixture was separated by column chromatography (eluent volume ratio: petroleum ether:ethyl acetate = 3:1) to obtain 0.30 g of a white solid product A4, the structural formula of which is shown below, with a yield of 73.1%.

[0076]

[0077] (2) A4 (0.30 g, 0.48 mmol) was dissolved in 2 mL of dichloromethane, and trifluoroacetic acid (0.55 g, 4.8 mmol) was slowly added dropwise under ice bath conditions. After the addition was complete, the temperature was raised to room temperature and kept warm for 6 h. The reaction progress was monitored by plate counting. After the reaction was completed, the solvent was evaporated by vacuum distillation, and 3 mL of dichloromethane was added and the mixture was dried again. The above operation was repeated 5 times until all the trifluoroacetic acid was removed. The mixture was dried under vacuum to obtain 0.28 g of a white solid product B4, i.e., the cationic lipid compound in this example, with a yield of 89.2%. The characterization results of B4 were as follows:

[0078] 1 H NMR: (400MHz, CDCl3): δ(ppm)5.04(m,1H,-COOCH-),3.43(t,2H,-CH2NHCH2-),3.27(m,2H,-CH2NHCH2-),3.08(m,2H,-CH2CH2NHCH2 CH2-),2.75(m,2H,-CH2CH2NHCH2CH2-,-CHCOO-),1.81(m,2H,-COOCHCH2-),1.25(m,42H,-CH2CH2-,-CH2CH3),0.88(t,6H,CH3CH2-)

[0079] HR-MS: [M+H] + :537.4996

[0080] Example 5

[0081] A cationic lipid compound, the structural formula of which is shown below:

[0082]

[0083] The cationic lipid compound in this embodiment was prepared by the following steps:

[0084] (1) N-Boc-3-(methylamino)propionic acid (0.15 g, 0.74 mmol), tetradecanal (0.16 g, 0.74 mmol) and dodecanonitrile (0.14 g, 0.74 mmol) were dissolved in 2 mL of tetrahydrofuran, heated to 45°C and kept warm for 24 h. The solvent was then removed by distillation under reduced pressure, and the product A5 was separated by column chromatography (eluent volume ratio: petroleum ether:ethyl acetate = 3:1) to obtain 0.38 g of a white solid product A5, the structural formula of which is as follows, with a yield of 83.2%:

[0085]

[0086] (2) A5 (0.38 g, 0.62 mmol) was dissolved in 2 mL of dichloromethane, and trifluoroacetic acid (0.71 g, 6.2 mmol) was slowly added dropwise under ice bath conditions. After the addition was complete, the temperature was raised to room temperature and kept warm for 6 h. The reaction progress was monitored by plate counting. After the reaction was completed, the solvent was evaporated by vacuum distillation, and 3 mL of dichloromethane was added and the mixture was dried again. The above operation was repeated 5 times until all the trifluoroacetic acid was removed. The mixture was dried under vacuum to obtain 0.36 g of a white solid product B5, i.e., the cationic lipid compound in this example, with a yield of 92%. The characterization results of B5 were as follows:

[0087] 1 H NMR: δ(ppm)5.08(m,1H,-COOCH-),3.39(d,3H,-CH3NH-),3.25(m,2H,-CONHCH2-),2.80(m,4H,CH3NHCH2CH2-,CH3NHCH2CH2-) ,2.75(m,2H,-CH2CH2NHCH2CH2-,-CHCOO-),1.81(m,2H,-COOCHCH2-),1.25(m,42H,-CH2CH2-,-CH2CH3),0.88(t,6H,CH3CH2-)

[0088] HR-MS: [M+H] + :511.4821

[0089] Experimental example

[0090] 1. Preparation of LNP:

[0091] The cationic lipid compounds B1 and B2 (0.0025 mmol) and DOPE (0.005 mmol) prepared in Examples 1 to 2 were weighed separately and added to a 10 mL ground-mouth round-bottom test tube. 1.5 mL of anhydrous chloroform was added to dissolve the mixture. The solvent was slowly dried at room temperature using a rotary evaporator (12 r / min) to obtain a thin film attached to the wall of the test tube, which was vacuum-dried overnight. 2.5 mL of ultrapure water was added, and the mixture was heated in a 70°C water bath for 30 min to detach the liposome membrane. The mixture was vortex-mixed and pulverized in an ultrasonic grinder for 10 min in an ice bath to obtain a 1 mmol / L LNP solution, which was stored in a 4°C refrigerator for later use.

[0092] Cationic lipid molecules B3, B4 and B5 (0.0025 mmol) and DOPE (0.0025 mmol) prepared in Examples 3 to 5 were weighed separately and added to a 10 mL ground-mouth round-bottom test tube. 1.5 mL of anhydrous chloroform was added to dissolve the mixture. The solvent was slowly dried using a rotary evaporator (12 rpm) at room temperature to obtain a thin film attached to the wall of the test tube, which was then vacuum-dried overnight. 2.5 mL of ultrapure water was added, and the mixture was heated in a 70°C water bath for 30 min to detach the liposome membrane. The mixture was vortex-mixed and pulverized in an ultrasonic grinder for 10 min under ice bath conditions to obtain a 1 mmol / L LNP solution, which was stored in a 4°C refrigerator for later use.

[0093] 2. Investigating the gene delivery performance of LNP / plasmid complexes

[0094] In order to investigate the gene delivery performance of the LNPs (B1, B2, B3, B4, B5) obtained in the above application examples, the following tests were performed:

[0095] (1) Particle size and zeta potential of LNP

[0096] Take 20 μL of the prepared LNP solution (1 mmol / L), dilute it with ultrapure water to a concentration of 20 nmol / mL, and the total volume of the solution is 1 mL. Mix it evenly by pipetting. Use a nanoparticle size and potential analyzer ZEN 3600 (produced by Malvern Inc.) to measure the particle size and Zeta potential. The results are as follows: Figure 2 As shown, Figure 2 A in the equation is the particle size of LNP, Figure 2 B represents the zeta potential of the LNP. As can be seen from the figure, the particle sizes of B1, B2, B3, B4, and B5 are all between 100 and 200 nm, and their potentials are positive, ranging from 20 to 50 mV. Preliminary results indicate that these properties meet the requirements for gene delivery and can bind to DNA.

[0097] (2) Gel electrophoresis experiment of LNP / pUC-19 plasmid complex

[0098] The prepared LNP solution (1 mmol / L) was mixed evenly with pUC-19 plasmid (0.5 mg / mL) at N / P ratios of 0, 1, 2, 4, and 6, respectively. The mixture was then diluted with ultrapure water to a pUC-19 plasmid concentration of 12.5 μg / mL and a total volume of 20 μL. The mixture was then pipetted and mixed evenly. The mixture was incubated at room temperature for 30 min to obtain a complex of LNP and pUC-19 plasmid.

[0099] Add 1.2g of agarose (Amor, USA) to a 250mL Erlenmeyer flask, then add 120mL of TAE buffer (prepared from analytically pure chemical reagents). Heat to completely dissolve the agarose particles, yielding a colorless, transparent liquid. Cool to 50°C-60°C, add 2.5μL of GelRed nucleic acid dye, mix thoroughly, and slowly pour into a pre-adjusted horizontal gel casting trough equipped with a comb. Allow to stand at room temperature until the gel is completely solidified, then carefully remove the comb to obtain a 1% agarose gel. Prepared LNP / pUC-19 plasmid complexes with varying nitrogen-phosphorus ratios were added to the wells of the gel. 0.125μg of pUC-19 plasmid served as a blank control. 2.5μL of 10× loading buffer (Shanghai Baili Biotechnology Co., Ltd.) was added to each well to fix the material. Then place the gel-making tank with the gel into the electrophoresis tank, add TEA buffer, and cover the electrophoresis tank. Run the electrophoresis at 150V for about 15 minutes at room temperature (25°C). Remove the gel and expose it in the gel imaging system GelDoc 2000 (manufactured by BIO-RAD, USA) to collect images. The results are as follows: Figure 3 As shown in the figure, B1 and B2 completely block DNA at N / P = 2, while B3, B4, and B5 completely block DNA at N / P = 4. All LNPs achieve DNA encapsulation at relatively low nitrogen-to-phosphorus ratios, exhibiting excellent plasmid binding capabilities and potential for gene delivery.

[0100] (3) Cytotoxicity experiments of B1 / pGL-3 plasmid complex and B2 / pGL-3 plasmid complex

[0101] HepG2 cells were seeded in 96-well plates (produced by Corning, USA) at a density of 10 4 Cells were cultured at 400 μL / well in 100 μL DMEM complete medium containing 10% fetal bovine serum (FBS) and 1% double-antibody (penicillin + streptomycin, 10,000 U / mL) and incubated in an incubator (manufactured by JOUAN, France) at 37°C and 5% CO2 for 24 h to allow the cell growth density to reach 70-80%.

[0102] The prepared LNP solutions (B1 and B2) (1 mmol / L) were mixed with pGL-3 plasmid (1 mg / mL) at N / P ratios of 1, 2, 4, 6, 8, and 10, then diluted with Opti-MEM medium to a pGL-3 plasmid concentration of 4 μg / mL in a total volume of 50 μL. Five replicate wells were set for each ratio. A pGL-3 plasmid without LNP was used as a blank control, and a Lipo 2000 / pGL-3 plasmid complex was used as a positive control. The LNP / pUC-19 plasmid complex was obtained by incubation at room temperature for 30 minutes.

[0103] Discard the existing culture medium in the wells and add 50 μL of complete DMEM medium to each well. Add 50 μL of the pre-prepared LNP / pGL-3 plasmid complex solution to each well. Continue incubating at 37°C, 5% CO2 for 24 hours. Remove the old culture medium and add 100 μL of serum-free and antibiotic-free DMEM medium (containing 10 μL of CCK-8 solution) to each well. Incubate for 30 minutes. Measure the absorbance of the solution at 450 nm using a microplate reader (Bio-RAD, USA). The relative cell viability formula is as follows:

[0104] Cell viability (%) = (OD 450 Sample / OD 450 Blank control) × 100%

[0105] Among them, OD is the average value measured by 5 parallel samples.

[0106] Test results such as Figure 4 As shown in the figure, the cell viability of B1 and B2 after 24 hours of transfection is generally above 75%, while the cell viability of commercially available Lipo 2000 is only 57% when the nitrogen-phosphorus ratio achieves the optimal transfection efficiency. B1 and B2 are less cytotoxic than commercially available Lipo 2000 and have potential development value.

[0107] (4) Cytotoxicity experiments of B3 / pGL-3 plasmid complex, B4 / pGL-3 plasmid complex, and B5 / pGL-3 plasmid complex

[0108] A549 cells were seeded in a 96-well plate (produced by Corning, USA) at a density of 10 4 Cells were cultured at 4% 4% 4% CO2 per well in 100 μL 1640 complete medium containing 10% fetal bovine serum (FBS) and 1% double-antibody (penicillin + streptomycin, 10,000 U / mL) in an incubator (manufactured by JOUAN, France) at 37°C and 5% CO2 for 24 h to allow the cell growth density to reach 70-80%.

[0109] The prepared LNP solutions (B3, B4, and B5) (1 mmol / L) were mixed evenly with pGL-3 plasmid (1 mg / mL) at N / P ratios of 1, 2, 4, 6, 8, and 10, then diluted with Opti-MEM medium to a pGL-3 plasmid concentration of 4 μg / mL in a total volume of 50 μL. Five replicate wells were set for each ratio. A pGL-3 plasmid without LNP was used as a blank control, and a Lipo 2000 / pGL-3 plasmid complex was used as a positive control. The LNP / pUC-19 plasmid complex was obtained by incubation at room temperature for 30 minutes.

[0110] Discard the existing culture medium in each well and add 50 μL of 1640 complete medium to each well. Add 50 μL of the pre-prepared LNP / pGL-3 plasmid complex solution to each well. Continue incubating at 37°C, 5% CO2 for 24 hours. Remove the old culture medium and add 100 μL of serum-free and antibiotic-free 1640 medium (containing 10 μL of CCK-8 solution) to each well. Incubate for 30 minutes. Measure the absorbance of the solution at 450 nm using a microplate reader (Bio-RAD, USA). The relative cell viability formula is as follows:

[0111] Cell viability (%) = (OD 450 Sample / OD 450 Blank control) × 100%

[0112] Among them, OD is the average value measured by 5 parallel samples.

[0113] Test results such as Figure 5 As shown in the figure, cell viability of B3 and B5 reached over 95% 24 hours after transfection, and that of B4 reached 60% 24 hours after transfection. Commercially available Lipo 2000 achieved a cell viability of approximately 91% at the nitrogen-phosphorus ratio that optimizes transfection efficiency. B3 and B5 exhibit lower cytotoxicity than Lipo 2000 and therefore have greater development value.

[0114] (5) In vitro qualitative transfection experiments of B1 / pEGFP-N1 plasmid complexes and B2 / pEGFP-N1 plasmid complexes

[0115] HepG2 cells were seeded in 48-well plates (produced by Corning, USA) at a density of 5×10 4 Cells were cultured at 400 μL / well in 250 μL DMEM complete medium containing 10% fetal bovine serum (FBS) and 1% double-antibody (penicillin + streptomycin, 10,000 U / mL) and incubated in an incubator (manufactured by JOUAN, France) at 37°C and 5% CO2 for 24 h to allow the cell growth density to reach 70-80%.

[0116] The prepared LNP solutions (B1 and B2) (1 mmol / L) were mixed with pEGFP-N1 plasmid (1 mg / mL) at N / P ratios of 1, 2, 4, 6, 8, and 10, respectively. The mixture was then diluted with Opti-MEM medium to a pEGFP-N1 plasmid concentration of 8 μg / mL in a total volume of 50 μL. A Lipo 2000 / pEGFP-N1 plasmid complex was used as a positive control. The LNP / pEGFP-N1 plasmid complex was obtained by incubation at room temperature for 30 minutes.

[0117] Discard the original culture medium in the wells, add 200 μL of DMEM complete medium with or without serum to each well, add 50 μL of the pre-prepared LNP / pEGFP-N1 plasmid complex solution, and continue to culture at 37°C, 5% CO2 for 4 hours. After that, remove the old culture medium and add 250 μL of DMEM serum medium to each well. Continue to culture in the incubator for 20 hours. Then take out the cell plate and observe the expression of pEGFP protein under an inverted fluorescence microscope (produced by Olympus, Japan) and take pictures. The results are shown in Figure 2. Figure 6 As shown in the figure, B1 and B2 have a certain transfection effect in HepG cells, but the transfection effect is lower than that of Lipo2000.

[0118] (6) In vitro transfection experiments of B3 / pEGFP-N1 plasmid complex, B4 / pEGFP-N1 plasmid complex and B5 / pEGFP-N1 plasmid complex:

[0119] A549 cells were seeded in 48-well plates (produced by Corning, USA) at a density of 5×10 4 Cells were cultured at 400 μg / well in 250 μL 1640 complete medium containing 10% fetal bovine serum (FBS) and 1% double-antibody (penicillin + streptomycin, 10,000 U / mL) and incubated in an incubator (manufactured by JOUAN, France) at 37°C and 5% CO2 for 24 h to allow the cell growth density to reach 70-80%.

[0120] The prepared LNP solutions (B3, B4, and B5) (1 mmol / L) were mixed with pEGFP-N1 plasmid (1 mg / mL) at N / P ratios of 1, 2, 4, 6, 8, and 10, respectively. The mixture was then diluted with Opti-MEM medium to a pEGFP-N1 plasmid concentration of 8 μg / mL in a total volume of 50 μL. A Lipo 2000 / pEGFP-N1 plasmid complex was used as a positive control. The LNP / pEGFP-N1 plasmid complex was obtained by incubation at room temperature for 30 minutes.

[0121] Discard the original culture medium in the wells, add 200 μL of 1640 complete medium with or without serum to each well, add 50 μL of the pre-prepared LNP / pEGFP-N1 plasmid complex solution, and continue to culture at 37°C, 5% CO2 for 4 hours. After that, remove the old culture medium and add 250 μL of 1640 serum medium to each well. Continue to culture in the incubator for 20 hours. Then remove the cell plate and observe the expression of pEGFP protein under an inverted fluorescence microscope (produced by Olympus, Japan) and take pictures. The results are shown in Figure 2. Figure 7 As shown in the figure, B3, B4, and B5 all have good transfection effects in A549, and their transfection effects are better than Lipo2000.

[0122] (7) In vitro quantitative transfection experiments of B1 / pGL-3 plasmid complexes and B2 / pGL-3 plasmid complexes

[0123] HepG2 cells were seeded in 48-well plates (produced by Corning, USA) at a density of 5×10 4 Cells were cultured at 400 μL / well in 250 μL DMEM complete medium containing 10% fetal bovine serum (FBS) and 1% double-antibody (penicillin + streptomycin, 10,000 U / mL) and incubated in an incubator (manufactured by JOUAN, France) at 37°C and 5% CO2 for 24 h to allow the cell growth density to reach 70-80%.

[0124] The prepared LNP solutions (B1 and B2) (1 mmol / L) were mixed with pGL-3 plasmid (1 mg / mL) at N / P ratios of 1, 2, 4, 6, 8, and 10, respectively. The mixture was then diluted with Opti-MEM medium to a pGL-3 plasmid concentration of 8 μg / mL in a total volume of 50 μL. Three replicate wells were set for each ratio. Lipo2000 / pGL-3 plasmid complexes served as positive controls. The LNP / pGL-3 plasmid complexes were obtained by incubation at room temperature for 30 min.

[0125] The original culture medium in the wells was discarded, and 200 μL of serum-containing or serum-free DMEM complete medium was added to each well. 50 μL of the pre-prepared LNP / pGL-3 plasmid complex solution was added to each well. After incubation at 37°C and 5% CO2 for 4 hours, the old culture medium was removed, and 250 μL of serum-containing DMEM medium was added to each well. The cells were incubated in an incubator for another 20 hours. The culture medium was aspirated, and each well was washed twice with 200 μL of pre-chilled PBS buffer (1×). According to the protocol provided by the manufacturer of the luciferase kit (Promega), 60 μL (1× concentration) of cell lysis buffer was added to the cells. After lysis at room temperature for 30 minutes, the cell lysate was transferred to a 1.5 mL EP tube and centrifuged at 12,000 rpm for 10 minutes. 20 μL of the supernatant was collected and transferred to a 96-well plate. Then, 100 μL of luciferase substrate was added to allow them to complex, and the fluorescence activity was measured using a microplate reader (manufactured by Thermo Corporation, USA). The protein concentration of the lysate was determined using the Thermo Modified Lowry Protein Assay (Thermo, Rockford, IL, USA). The transfection efficiency was expressed as relative fluorescence activity (RLU) / mg protein, i.e., RLU / mg protein. The transfection results were the average of three parallel experiments.

[0126] The results are as follows Figure 8 As shown in the figure, although the transfection effect of B1 and B2 is slightly worse than that of Lipo 2000, it has reached 10 7 , which has the possibility of clinical application.

[0127] (8) In vitro quantitative transfection experiments of B3 / pGL-3 plasmid complexes, B4 / pGL-3 plasmid complexes, and B5 / pGL-3 plasmid complexes

[0128] A549 cells were seeded in 48-well plates (produced by Corning, USA) at a density of 5×10 4 Cells were cultured at 400 μL / well in 250 μL 1640 complete medium containing 10% fetal bovine serum (FBS) and 1% double-antibody (penicillin + streptomycin, 10,000 U / mL) and incubated in an incubator (manufactured by JOUAN, France) at 37°C and 5% CO2 for 24 h to allow the cell growth density to reach 70-80%.

[0129] The prepared LNP solutions (B3, B4, and B5) (1 mmol / L) were mixed with pGL-3 plasmid (1 mg / mL) at N / P ratios of 1, 2, 4, 6, 8, and 10, respectively. The mixture was then diluted with Opti-MEM medium to a pGL-3 plasmid concentration of 8 μg / mL in a total volume of 50 μL. Three replicate wells were set for each ratio. Lipo2000 / pGL-3 plasmid complexes served as positive controls. The LNP / pGL-3 plasmid complexes were obtained by incubation at room temperature for 30 minutes.

[0130] The original medium in the wells was discarded, and 200 μL of serum-containing or serum-free 1640 complete medium was added to each well. 50 μL of the pre-prepared LNP / pGL-3 plasmid complex solution was added to each well. After incubation at 37°C, 5% CO2 for 4 h, the old medium was removed, and 250 μL of serum-containing 1640 medium was added to each well. The cells were incubated in an incubator for another 20 h. The medium was aspirated, and each well was washed twice with 200 μL of pre-chilled PBS buffer (1×). According to the protocol provided by the manufacturer of the luciferase kit (Promega), 60 μL (1× concentration) of cell lysis buffer was added to the cells. After lysis at room temperature for 30 min, the cell lysate was transferred to a 1.5 mL EP tube and centrifuged at 12,000 rpm for 10 min. 20 μL of the supernatant was transferred to a 96-well plate. Then, 100 μL of luciferase substrate was added to allow them to complex, and the fluorescence activity was measured using a microplate reader (manufactured by Thermo Corporation, USA). The protein concentration of the lysate was determined using the Thermo Modified Lowry Protein Assay (Thermo, Rockford, IL, USA). The transfection efficiency was expressed as relative fluorescence activity (RLU) / mg protein, i.e., RLU / mg protein. The transfection results were the average of three parallel experiments.

[0131] The results are as follows Figure 9 As shown in the figure, the transfection effects of B3, B4, and B5 in A549 cells were 10 to 20 times that of Lipo 2000 in both the presence and absence of serum, which is superior to Lipo 2000 and has the potential for clinical application.

[0132] Although the specific embodiments of the present invention have been described in detail in conjunction with the embodiments, this should not be construed as limiting the scope of protection of this patent. Within the scope described by the claims, various modifications and variations that can be made by those skilled in the art without creative work still fall within the scope of protection of this patent.

Claims

1. A cationic lipid compound, characterized in that The cationic lipid compound is one of the following compounds: 、 、 、 、 。 2. The method for preparing the cationic lipid compound according to claim 1, wherein The following steps are involved: S1: dissolving the acid, aldehyde, and isonitrile in a first organic solvent, reacting at 40-50°C for 16-32 hours, then removing the solvent and purifying to obtain product A; The acid is one of the following acids: The aldehyde is one of the following aldehydes: The isonitrile is one of the following isonitriles: S2: Dissolve product A in a second organic solvent, add trifluoroacetic acid under ice bath conditions, then warm to room temperature and keep warm for 5-8 hours; then spin dry the solvent and remove the trifluoroacetic acid to obtain the product.

3. The preparation method according to claim 2, wherein: The molar ratio of the acid, aldehyde and isonitrile is 1:1:

1.

4. The preparation method according to claim 2, wherein: The first organic solvent is tetrahydrofuran; the second organic solvent is dichloromethane.

5. The preparation method according to claim 2, wherein: The purification method in S1 is column chromatography, and the eluent used in the column chromatography is obtained by mixing petroleum ether and ethyl acetate in a volume ratio of 3:

1.

6. The preparation method according to claim 2, characterized in that The method for removing trifluoroacetic acid in S2 is: dissolving the substance after the solvent is spin-dried in a second organic solvent, and then spin-dried; repeating the above operation 4 to 6 times.

7. Use of the cationic lipid compound according to claim 1 in preparing a gene carrier.

8. The use according to claim 7, characterized in that The gene carrier is prepared by the following steps: adding a cationic lipid compound and DOPE in a molar ratio of 1:1-2 into a reaction vessel, dissolving them with anhydrous chloroform, then spinning off the solvent to obtain a thin film attached to the side wall of the reaction vessel, and vacuum drying overnight; then adding ultrapure water to the reaction vessel, heating at 70°C for 30 minutes to cause the thin film to fall off, and then crushing in an ice bath to obtain a gene carrier solution.

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