A siRNA delivery system and its preparation method, application and product

Abstract

The present invention belongs to the field of biomedicine, and specifically relates to a siRNA delivery system and a preparation method, application and product thereof. The present invention provides a siRNA delivery system, wherein the siRNA delivery system is a lipid nanoparticle; the lipid nanoparticle includes: 4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester mc3, ((4-hydroxybutyl) azadialkyl) bis(hexane-6,1-diyl) bis(2-hexyldecanoate), 1,2-distearoylphosphatidylcholine, pegylated lipid and cholesterol; the molecular weight of the pegylated lipid is 2509.2. The lipid nanoparticles provided by the present invention have a high delivery efficiency, a high efficiency of suppressing target genes, and good dispersibility of lipid nanoparticles and high safety.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a siRNA delivery system and a preparation method, application and product thereof. Background Art

[0002] Common wrinkles are caused by muscle traction or morphological changes. The target gene SNAP-25 is one of the three proteins in the SNARE (Soluble N-ethylmaie-sensitive factor attachment protein receptors, SNARE, soluble N-ethylmaleimide sensitive factor attachment protein receptor) complex, and is also the main protein for the decomposition of botulinum toxin. In nerve cells, SNAP-25 affects axon growth and synaptic plasticity, and can regulate neurotransmitter release. The light chain of botulinum toxin type A protein can specifically hydrolyze the SNAP-25 protein (synaptosome-associated protein-25) on the presynaptic membrane, thereby affecting the activity of SNARE proteins involved in cell endocytosis and exocytosis, resulting in the inability of acetylcholine-containing vesicles to bind to the plasma membrane of the presynaptic membrane, and thus cannot be released. The corresponding nerve impulses cannot stimulate the contraction of the muscles they control, so it can improve facial dynamic wrinkles, and its effect is reversible.

[0003] SiRNA is a double-stranded RNA molecule, usually 21-23 nucleotides in length, with a specific sequence that can fully or partially complement the mRNA of the target gene. In cells, SiRNA is recognized and bound by the RNA-induced silencing complex (RISC), which then uses SiRNA as a guide to cut the complementary mRNA molecule, thereby blocking the translation process of the target gene and achieving the purpose of inhibiting gene expression. However, the low transmission efficiency of SiRNA (small interfering RNA) has resulted in its unsatisfactory inhibitory effect on the gene SNAP-25.

[0004] Therefore, it is necessary to develop a SiRNA delivery system to improve the SiRNA delivery efficiency and thereby achieve a better inhibitory effect on the gene SNAP-25. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a siRNA delivery system and a preparation method, application and product thereof.

[0006] On the one hand, the present invention provides a siRNA delivery system, which is a lipid nanoparticle; the lipid nanoparticle comprises: 4-(N,N-dimethylamino)butyric acid (dialene) methyl ester mc3, ((4-hydroxybutyl) azadialkyl) bis(hexane-6,1-diyl) bis(2-hexyldecanoate), 1,2-distearoylphosphatidylcholine, pegylated lipid and cholesterol; the molecular weight of the pegylated lipid is 2509.2.

[0007] Specifically, in the lipid nanoparticles, the molar ratios of 4-(N,N-dimethylamino)butyric acid (dialene) methyl ester mc3, ((4-hydroxybutyl) azadialkyl) bis(hexane-6,1-diyl) bis(2-hexyldecanoate), 1,2-distearoylphosphatidylcholine, PEGylated lipids and cholesterol are (35-40):(10-15):(10-15):(1-3):(35-40).

[0008] Furthermore, in the lipid nanoparticles, the molar ratio of 4-(N,N-dimethylamino)butyric acid (dialene) methyl ester mc3, ((4-hydroxybutyl) azadialkyl) bis(hexane-6,1-diyl) bis(2-hexyldecanoate), 1,2-distearoylphosphatidylcholine, PEGylated lipid and cholesterol is (35-40): (10-15): 10: 1.5: 38.5.

[0009] Furthermore, in the lipid nanoparticles, the molar ratio of 4-(N,N-dimethylamino)butyric acid (dialene) methyl ester mc3, ((4-hydroxybutyl) azadialkyl) bis(hexane-6,1-diyl) bis(2-hexyldecanoate), 1,2-distearoylphosphatidylcholine, PEGylated lipid and cholesterol is 40:10:10:1.5:38.5 or 35:15:10:1.5:38.5.

[0010] Furthermore, in the lipid nanoparticles, the molar ratio of 4-(N,N-dimethylamino)butyric acid (dialene) methyl ester mc3, ((4-hydroxybutyl) azadialkyl) bis(hexane-6,1-diyl) bis(2-hexyldecanoate), 1,2-distearoylphosphatidylcholine, PEGylated lipid and cholesterol is 40:10:10:1.5:38.5.

[0011] Specifically, the molecular weights of the 4-(N,N-dimethylamino)butyric acid (dialene) methyl ester mc3, ((4-hydroxybutyl) azadialkyl) bis(hexane-6,1-diyl) bis(2-hexyldecanoate), 1,2-distearoylphosphatidylcholine and cholesterol are 642.09, 766.27, 790.15 and 386.66, respectively.

[0012] Specifically, the lipid nanoparticles also include siRNA.

[0013] In another aspect, the present invention provides a method for preparing the above-mentioned siRNA delivery system, comprising the following steps:

[0014] S1. preparing lipid-ethanol solution: dissolving 4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester mc3, ((4-hydroxybutyl) azadialkyl) bis(hexane-6,1-diyl) bis(2-hexyldecanoate), 1,2-distearoylphosphatidylcholine, PEGylated lipid and cholesterol in ethanol to prepare lipid-ethanol solution;

[0015] S2. Prepare nucleic acid-buffer solution: dissolve siRNA in acidic buffer;

[0016] S3, mixing and collecting: the lipid-ethanol solution of step S1 and the siRNA solution of step S2 are fully mixed to form lipid nanoparticles, and the lipid nanoparticles flowing out are collected.

[0017] Specifically, the ethanol in step S1 is anhydrous ethanol.

[0018] Specifically, the mass volume ratio of the five components to ethanol in step S1 is (6-8) mg / ml.

[0019] Furthermore, in step S1, the mass volume ratio of the five components to ethanol is 7.5 mg / ml.

[0020] Specifically, the preparation method further comprises:

[0021] S4, post-processing: removing the residual ethanol in the lipid nanoparticles collected in step S3;

[0022] S5. Quality detection of lipid nanoparticles: Perform quality detection on the lipid nanoparticles treated in step S4.

[0023] Specifically, the acidic buffer in step S2 is selected from one or more of Tris-HCl buffer, acetate buffer, phosphate buffer, tartaric acid buffer, succinate buffer, and HEPES buffer.

[0024] Furthermore, the acidic buffer in step S2 is Tris-HCl buffer.

[0025] Specifically, the volume concentration of the lipid-ethanol solution in step S3 is 15-30%.

[0026] Furthermore, the volume concentration of the lipid-ethanol solution in step S3 is 20%.

[0027] Specifically, the concentration of the siRNA in step S3 is 300-350ug / ml.

[0028] Furthermore, the concentration of the siRNA in step S3 is 330ug / ml.

[0029] Specifically, the mixing in step S3 adopts microfluidic mixing technology.

[0030] The liposome nanoparticle synthesis system requires a flow control system and a microfluidic chip, specifically including a 2-channel microinjection pump, 2 flow sensors, 2 liquid storage tubes and a microfluidic chip.

[0031] Specifically, the removal method in step S4 includes dialysis or ultrafiltration.

[0032] Specifically, the lipid nanoparticles in step S5 have a particle size of 80-90 mm;

[0033] Furthermore, the particle size of the lipid nanoparticles in step S5 is 88.41 mm.

[0034] In another aspect, the present invention provides use of the above siRNA delivery system or the siRNA delivery system prepared by the above preparation method in the preparation of a gene SNAP-25 inhibitor.

[0035] In another aspect, the present invention provides use of the above siRNA delivery system or the siRNA delivery system prepared by the above preparation method in the preparation of anti-wrinkle products.

[0036] In yet another aspect, the present invention provides an inhibitor, wherein the inhibitor comprises the above-mentioned siRNA delivery system.

[0037] In yet another aspect, the present invention provides an anti-wrinkle product, comprising the above-mentioned siRNA delivery system.

[0038] The technical effects achieved by the present invention are:

[0039] (1) The lipid nanoparticles provided by the present invention have a high delivery efficiency of up to 90%.

[0040] (2) The lipid nanoparticles provided by the present invention have a high inhibition efficiency on the target gene, which is 64.19% in SH-SY5Y cells and 61.13% in SK-N-MC cells.

[0041] (3) The lipid nanoparticles provided by the present invention have good dispersibility and high safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 The lipid nanoparticle delivery efficiency of Example 1 in SH-SY5Y cells.

[0043] Figure 2 is the lipid nanoparticle delivery efficiency of Example 1 in SK-N-MC cells.

[0044] Figure 3 Lipid nanoparticle delivery efficiency of Lipo3000 in SH-SY5Y cells.

[0045] Figure 4 Lipo3000 lipid nanoparticle delivery efficiency in SK-N-MC cells.

[0046] Figure 5 The lipid nanoparticle delivery efficiency of Example 2 in SH-SY5Y cells.

[0047] Figure 6 is the lipid nanoparticle delivery efficiency of Example 2 in SK-N-MC cells.

[0048] Figure 7 is the lipid nanoparticle delivery efficiency of Comparative Example 1 in SH-SY5Y cells.

[0049] Figure 8 is the lipid nanoparticle delivery efficiency of Comparative Example 1 in SK-N-MC cells.

[0050] Fig. 9 is the lipid nanoparticle delivery efficiency of Comparative Example 2 in SH-SY5Y cells.

[0051] Fig.10 is the lipid nanoparticle delivery efficiency of Comparative Example 2 in SK-N-MC cells.

[0052] Fig.11 is the lipid nanoparticle delivery efficiency of Comparative Example 3 in SH-SY5Y cells.

[0053] Fig.12 is the lipid nanoparticle delivery efficiency of Comparative Example 3 in SK-N-MC cells.

[0054] Fig.13 is the lipid nanoparticle delivery efficiency of Comparative Example 4 in SH-SY5Y cells.

[0055] Fig.14 is the lipid nanoparticle delivery efficiency of Comparative Example 4 in SK-N-MC cells.

[0056] Fig.15 is the inhibition rate of SNAP25 expression in SH-SY5Y cells.

[0057] Fig.16is the inhibition rate of SNAP25 expression in SH-SY5Y cells.

[0058] Fig.17 is the inhibition rate of SNAP25 expression in SK-N-MC cells.

[0059] Fig.18 is the inhibition rate of SNAP25 expression in SK-N-MC cells.

[0060] Fig.19 The inhibition efficiency of the target gene in SK-N-MC cells is shown in the figure. A is the control group, B is the NC group, C is the low-dose group, D is the medium-dose group, and E is the high-dose group.

[0061] Fig. 20 The inhibition efficiency of the target gene in SK-N-MC cells is shown in the figure. A is the control group, B is the NC group, C is the low-dose group, D is the medium-dose group, and E is the high-dose group. DETAILED DESCRIPTION

[0062] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0063] The main reagents, cells and antibodies of the present invention are shown in Table 1:

[0064] Table 1

[0065]

[0066] Example 1

[0067] The delivery materials tested in the present invention are as follows:

[0068] (1) Lipid Nanoparticles

[0069] The ingredients of the lipid nanoparticles of the present invention are shown in Table 2:

[0070] Table 2

[0071]

[0072] Synthesis method: The liposome nanoparticle synthesis system requires a flow control system and a microfluidic chip, specifically including a 2-channel microinjection pump, 2 flow sensors, 2 liquid storage tubes and a microfluidic chip.

[0073] The workflow of the liposome nanoparticle synthesis system is as follows: the pressure controller connected to the pressure source provides a stable pressure value, and a fixed pressure is applied to the sealed liquid storage tube through the pipeline, so that the liquid in the liquid storage tube is stably output and passes through the flow sensor. The flow sensor measures the flow and feeds back to the pressure controller, thereby achieving the purpose of precise flow control. The fluid is then stably and accurately delivered to the microfluidic chip, and the specific parameters can be controlled and set through the software on the computer. The 2-channel microinjection pump controls the lipid-containing ethanol phase and water phase solution respectively. The two-phase solution can adjust the flow rate separately, and mix in the microfluidic chip after passing through the flow sensor and damper, thereby synthesizing nanoliposomes. Specifically, nanoliposomes with a fixed particle size can be obtained by adjusting the flow rate, flow rate ratio and total flow rate of the ethanol phase and the water phase.

[0074] When siRNA encapsulation is performed, first, according to the above-mentioned lipid nanoparticle formula and ratio, the five lipid components are dissolved in anhydrous ethanol to prepare a lipid ethanol solution, and the mass volume ratio of the five components to ethanol is 7.5 mg / ml. At the same time, the siRNA is dissolved in an acidic buffer (Tris-HCl in the present invention) to ensure that the siRNA is in a form suitable for binding to the lipid. The lipid-ethanol solution and the siRNA solution are fully mixed in a micromixer using a microfluidic chip, the volume concentration of the lipid-ethanol solution is 20%, and the concentration of the siRNA is 330ug / ml to form lipid nanoparticles.

[0075] The workflow of the liposome nanoparticle synthesis system is as follows: the pressure controller connected to the pressure source provides a stable pressure value, and a fixed pressure is applied to the sealed liquid storage tube through the pipeline, so that the liquid in the liquid storage tube is stably output and passes through the flow sensor. The flow sensor measures the flow and feeds back to the pressure controller, thereby achieving the purpose of precise flow control. The fluid is then stably and accurately delivered to the microfluidic chip, and the specific parameters can be controlled and set through the software on the computer. The 2-channel microinjection pump controls the lipid-containing ethanol phase and water phase solution respectively. The two-phase solution can adjust the flow rate separately, and mix in the microfluidic chip after passing through the flow sensor and damper, thereby synthesizing nanoliposomes. Specifically, nanoliposomes with a fixed particle size can be obtained by adjusting the flow rate, flow rate ratio and total flow rate of the ethanol phase and the water phase.

[0076] For example, when encapsulating siRNA, firstly, according to the above LNP formula and ratio, the four lipid components are dissolved in anhydrous ethanol to prepare a lipid ethanol solution. At the same time, the siRNA is dissolved in an acidic buffer (such as tris-hcl) to ensure that the siRNA is in a form suitable for binding with lipids. The lipid-ethanol solution and the siRNA solution are fully mixed in a micromixer using a microfluidic chip to form LNP. In this process, the lipid is protonated in ethanol to form a cation, which binds to the negatively charged siRNA and self-assembles to form LNP through electrostatic interaction.

[0077] The mixed LNP solution contains a high concentration of ethanol, and the residual ethanol needs to be removed by dialysis or ultrafiltration, and the solution system is replaced with a neutral buffer for subsequent biological experiments and long-term storage.

[0078] In this process, lipids are protonated in ethanol to form cations, which bind to negatively charged siRNA and self-assemble into lipid nanoparticles through electrostatic interactions.

[0079] The mixed lipid nanoparticle solution contains a high concentration of ethanol, and the residual ethanol needs to be removed by dialysis or ultrafiltration, and the solution system needs to be replaced with a neutral buffer for subsequent biological experiments and long-term storage.

[0080] Quality testing of lipid nanoparticles: Use dynamic light scattering instrument and other equipment to test the particle size, monodispersity, etc. of lipid nanoparticles to ensure that the quality of lipid nanoparticles meets the experimental requirements.

[0081] Storage of lipid nanoparticles: Lipid nanoparticles that have passed quality inspection are stored at -80℃ for a long time.

[0082] Material related parameters are shown in Table 3:

[0083] Table 3

[0084]

[0085] (2) Commercially available transfection reagent Lipo3000 as a positive control

[0086] The implementation plan process and grouping are as follows:

[0087] 1) Human neuroblastoma cells SH-SY5Y and human neuroepithelioma cells SK-N-MC were selected, expanded and cultured, and plated on 12-well plates. When the cell confluence reached 80%, transfection was performed, and the amount of siRNA used per well was 2ug (1OD≈33ug).

[0088] 2) Naked siRNA and siRNA encapsulated by lipid nanoparticles.

[0089] (3) LIPOFECTAMIN3000 was used as the transfection reagent control (transfection of naked siRNA), and siRNA after lipid nanoparticle delivery material was used as the experimental group (Si-SNAP25 was synthesized by Suzhou Gene Gene Co., Ltd.). Each group was divided into the following subgroups:

[0090] ①Blank control group

[0091] ②NC group

[0092] ③Si-SNAP25 low dose group

[0093] ④Si-SNAP25 medium dose group

[0094] ⑤Si-SNAP25 high dose group

[0095] ⑥Si-SNAP25-cy3 low dose group

[0096] ⑦Si-SNAP25-cy3 medium dose group

[0097] ⑧Si-SNAP25-cy3 high dose group

[0098] At 24h and 48h after transfection, groups ⑥, ⑦, and ⑧ were stained with 4,6-diamidino-2-phenylindole and observed and photographed under a fluorescence microscope. The proportion of fluorescence-positive cells reflects the delivery efficiency of different delivery systems and conditions.

[0099] For groups ①, ②, ③, ④, and ⑤, cell samples were collected using TRIZOL and RIPA, respectively, and total RNA and protein were extracted and stored in a -80°C refrigerator for subsequent testing.

[0100] (4) Total RNA and total protein were extracted from the collected cells, and real-time fluorescence quantitative PCR (real-time PCR) and Western blot (antibody Santa Cruz sc-20038) were performed to compare the differences in the inhibitory efficiency of siRNA on target genes in different delivery systems.

[0101] The specific implementation plan is as follows:

[0102] 1.1 Cell culture

[0103] (1) Cell recovery

[0104] Take out the cells frozen in liquid nitrogen, quickly put them into 37℃ water, stir constantly, transfer them to a new 5mL centrifuge tube after thawing, add 3mL complete medium (DMEM+10%FBS+1% double antibody), centrifuge at 3000rpm for 5min, discard the supernatant, resuspend the cell pellet with 4mL complete medium, and spread it in a 6cm cell culture dish at 37℃, 5%CO2 Culture was continued and the medium was changed every other day.

[0105] (2) Cell passaging

[0106] When the cells grow to 80-90% confluence, they are passaged. Discard the supernatant of the cells, rinse them with PBS buffer, add 0.05 (W / V)% trypsin to cover the bottom surface, and digest at room temperature for about 1 minute. Aspirate the trypsin, add complete culture medium to terminate digestion, transfer to a new culture dish at a ratio of 1:2, and return to the incubator for continued culture.

[0107] 1.2 Cell transfection and delivery efficiency observation

[0108] (1) Cell transfection

[0109] The cells were plated in a 12-well plate and transfected when the confluence reached 70-80%.

[0110] Since the siRNA concentrations contained in the various delivery materials were inconsistent, a uniform siRNA amount was used for conversion to facilitate comparison, that is, the cell transfection amount per well was 2ug siRNA.

[0111] The amount of each material used for transfection is shown in Table 4 below:

[0112] Table 4

[0113]

[0114] The lipid nanoparticle delivery material can be directly added to the cell culture medium according to the above volume ratio, mixed and then replaced with the medium.

[0115] Lipo3000 operates according to the instructions:

[0116] a. Cells were seeded to 70-90% confluence for transfection.

[0117] b. Dilute Lipofectamine®3000 reagent (2 tubes) with Opti-MEM medium and mix thoroughly.

[0118] c. Prepare a DNA premix by diluting the DNA in Opti-MEM medium, then add P3000™ reagent and mix thoroughly.

[0119] d. Add diluted DNA to each tube of diluted Lipofectamine® 3000 reagent (1:1 ratio).

[0120] e. Incubate at room temperature for 5 minutes.

[0121] f. Add DNA-liposome complex to cells, mix well, and return to the incubator to continue culturing.

[0122] (2) Observation of transfection efficiency

[0123] 24 and 48 hours after transfection, collect and fix the cells: remove the culture medium, wash once with PBS, add 4,6-diamidino-2-phenylindole at a final concentration of 10ug / ml for nucleus staining, and incubate at room temperature for 5 minutes. Discard the supernatant, wash twice with PBS, and observe under a fluorescence microscope and take photos. Transfection efficiency = number of cy3 fluorescence positive cells / total number of cells × 100%.

[0124] Results: When the lipid composition (4-(N,N-dimethylamino)butyric acid (dialene) methyl ester mc3: ((4-hydroxybutyl) azadialkyl) bis(hexane-6,1-diyl) bis(2-hexyldecanoate): 1,2-distearoylphosphatidylcholine: PEGylated lipid: cholesterol, molar ratio of 40:10:10:1.5:38.5), the delivery efficiency of lipid nanoparticle Example 1 was ≥90%.

[0125] 1.3 Real-time PCR detection

[0126] (1) Extraction of total RNA

[0127] a. Remove the culture medium from each 6-well plate, wash once with PBS, add 1 mL of Trizol, operate on ice, and let stand at room temperature for 5 minutes to lyse;

[0128] b. Add chloroform (1 / 5 of the volume of Trizol) and shake vigorously for 15 seconds until the solution is fully emulsified without phase separation, and let stand at room temperature for 5 minutes;

[0129] c. Centrifuge at 12000 g for 15 min at 4°C;

[0130] d. Carefully remove the centrifuge tube, aspirate the supernatant and transfer it to another RNase free EP tube;

[0131] e. Add an equal volume of isopropanol, mix thoroughly by inverting, and let stand at room temperature for 10 min;

[0132] f. Centrifuge at 12000 g for 10 min at 4°C and discard the supernatant;

[0133] g. Wash with 1 mL of 75% ethanol, centrifuge at 12000 g for 5 min at 4°C, and carefully discard the ethanol;

[0134] h. Dry at room temperature for 2-3 minutes, add 20 μL of sterile ethylene pyrocarbonate water to dissolve the precipitate, and tap the bottom of the EP tube with your fingertips until it is completely dissolved.

[0135] i. Determine the concentration of RNA sample. OD 260 / OD 280 =1.91, the extracted RNA is of high purity and has no protein and DNA residues.

[0136] (2) cDNA synthesis

[0137] a. Prepare the mixture in a 0.2 mL RNase free EP tube and operate on ice.

[0138] b. 37℃, 15min, 85℃, 5s, 4℃, 60min. Add 80μL enzyme-free water to dilute the obtained cDNA for later use. It can be directly used for the synthesis of the second-strand cDNA (2nd-Strand cDNA) or PCR amplification and stored at -20℃.

[0139] The reverse transcription reaction system is shown in Table 5:

[0140] Table 5 Reverse transcription reaction system

[0141]

[0142] (3) Quantitative PCR reaction

[0143] a. The designed primer sequences and analysis conditions are shown in Tables 6 and 7.

[0144] Table 6 Primer information

[0145]

[0146] Table 7 PCR reaction system

[0147]

[0148] b. Mix well and perform 3 replicate controls for each sample.

[0149] c. Set up PCR program

[0150] Stage 1: Pre-denaturation

[0151] 95℃ 30s.

[0152] Stage 2: PCR reaction

[0153] 95℃ 05s.

[0154] 60℃ 30s.

[0155] Number of cycles: 45.

[0156] Phase 3: Melting curve determination

[0157] 95℃ 15s.

[0158] 60℃ 60s.

[0159] 95℃ 15s.

[0160] d. Amplification test on the machine, using 2 –ΔΔCt The relative expression level was calculated.

[0161] The results are as follows Figure 15-18 As shown: By comparing the seven groups of experimental data, it was found that the cell conversion rate using LNP1 as a carrier to act on the target gene was the lowest, indicating that LNP1 had the best inhibitory effect, and the inhibitory effect increased with the increase of LNP1 concentration.

[0162] 1.4 Western-blot detection

[0163] Protein extraction and concentration determination

[0164] (1) Protein extraction

[0165] a. Take an appropriate amount of cells and wash them three times with ice-cold PBS.

[0166] b. Take an appropriate amount of lysate and add phenylmethylsulfonyl fluoride (PMSF) at a ratio of 1:100 within a few minutes before use.

[0167] c. Mix the cells by pipetting and place them in a refrigerator at 4 degrees for about 20 minutes.

[0168] d. After sufficient lysis, centrifuge at 11000r at 4 degrees for 10 minutes and take the supernatant.

[0169] (2) Protein quantification

[0170] a. According to the number of samples, prepare an appropriate amount of BCA working solution by adding 50 volumes of BCA reagent A to 1 volume of BCA reagent B (50:1) and mix thoroughly.

[0171] b. Configure the standard curve wells according to the BCA quantitative method, add 2 μL of sample to the ELISA plate, and fill up to 20 μL with PBS solution.

[0172] c. Add 200 ml of BCA working solution to each well and incubate at 37°C for 20 minutes until a color gradient change is visible.

[0173] d. Determine A562 and calculate the protein concentration based on the standard curve.

[0174] (3) SDS-PAGE electrophoresis

[0175] a. Prepare sample solution: Mix sample with 5X loading buffer at a ratio of 4:1 and boil for 10 minutes.

[0176] b. Load the prepared samples and protein marker (10-180KD) separately.

[0177] c. Electrophoresis: Using an electrophoresis device, the sample is first electrophoresed at a constant voltage of 80V until the dye approaches the top of the separation gel. Then, the sample is electrophoresed at a constant voltage of 120V until the bromophenol blue just exits the bottom of the gel.

[0178] (4) Protein transfer

[0179] a. Remove the gel and discard all the stacking gel. Soak the gel in transfer buffer for 5 minutes.

[0180] b. Prepare the membrane: Pre-immerse the NC membrane in transfer buffer for a few seconds.

[0181] c. Assemble the transfer sandwich in the order of foam, filter paper, gel, transfer membrane, filter paper, and foam.

[0182] d. Place the transfer clip into the transfer tank, making sure the gel side of the transfer clip faces the cathode (-) and the membrane side faces the anode (+). Add an appropriate amount of buffer to the transfer tank, making sure the transfer clip is completely submerged.

[0183] e. Place the transfer device in ice water and turn on the power at 350mA for 2 hours.

[0184] f. When the transfer is complete, remove the transfer clip from the tank, carefully open the transfer stack with tweezers, and rinse the membrane in double distilled water.

[0185] (5) Protein imaging

[0186] a. Place the membrane in the reaction chamber (protein-stained side facing up).

[0187] b. Add 0.5 mL of Ponceau red for 30 seconds and observe the transfer effect.

[0188] c. Remove the stain and wash the membrane three times with double distilled water, 5 minutes each time.

[0189] (6) Immunoassay

[0190] a. Blocking: Freshly prepare blocking solution: Add skim milk powder to TBST to a final concentration of 5% (w / v). When blocking, immerse the membrane in the blocking solution and oscillate at room temperature for 1 hour.

[0191] b. Add the diluted primary antibody (SNAP 25 antibody, purchased from SANTA CRUZ, catalog number sc-20038, dilution ratio 1:200) and incubate at 4°C overnight.

[0192] c. Wash the membrane three times with TBST (10 min each time).

[0193] d. Add the corresponding diluted secondary antibody (horseradish peroxidase-labeled goat anti-mouse IgG (H+L), product number A0216, purchased from Beyotime, dilution ratio 1:1000), and incubate with shaking at room temperature for 1 hour.

[0194] e. Wash the membrane three times with TBST (10 min each time).

[0195] (7) Chemiluminescence detection

[0196] a. After washing the membrane, take out the ECL luminescent reagent and mix solution A and solution B in a 1:1 ratio.

[0197] b. Place the membrane in a fluorescent chemiluminescence imager, add luminescent liquid, perform three exposures, and select the overlap value of the three exposures.

[0198] The results are as follows Figure 19-20 As shown: the cell conversion rate of the target gene with LNP1 as a carrier decreased significantly with the increase of dosage concentration, which was manifested as a decrease in protein conversion rate, a significant decrease in the color of the electrophoresis graph from A to E, and the best inhibition effect.

[0199] The results showed that the delivery efficiency of lipid composition (4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester MC3: ((4-hydroxybutyl) azadialkyl) bis(hexane-6,1-diyl) bis(2-hexyldecanoate): 1,2-distearoylphosphatidylcholine: PEGylated lipid: cholesterol was 40:10:10:1.5:38.5) was: lipid nanoparticle Example 1 ≥ 90%; the inhibition rates in SH-SY5Y cells and SK-N-MC cells were 64.19% and 61.13%, respectively ( Figure 1-Figure 2 ).

[0200] Lipo3000 operates according to the instructions:

[0201] a. Transfect cells when the confluence is 70-90%

[0202] b. Dilute Lipofectamine®3000 reagent (2 tubes) with Opti-MEM medium and mix thoroughly.

[0203] c. Prepare a DNA premix by diluting the DNA in Opti-MEM medium, then add P3000™ reagent and mix thoroughly.

[0204] d. Add diluted DNA to each tube of diluted Lipofectamine® 3000 reagent (1:1 ratio).

[0205] e. Incubate at room temperature for 5 minutes.

[0206] f. Add DNA-liposome complex to cells, mix well, and return to the incubator to continue culturing.

[0207] The results showed that the delivery efficiency of lipo300 as the control group was ≥85%, the inhibition efficiency of the target gene in SH-SY5Y cells was about 58.47%, and the inhibition efficiency of the target gene in SK-N-MC cells was about 50.21% ( Figure 3-Figure 4 ).

[0208] Example 2

[0209] The difference between Example 2 and Example 1 is that:

[0210] The ingredients of the lipid nanoparticles of the present invention are shown in Table 8:

[0211] Table 8

[0212]

[0213] The rest is the same as in Example 1.

[0214] The results showed that when the lipid composition (4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester MC3: ((4-hydroxybutyl) azadialkyl) bis(hexane-6,1-diyl) bis(2-hexyldecanoate): 1,2-distearoylphosphatidylcholine: PEGylated lipid: cholesterol, the molar ratio was 35:15:10:1.5:38.5): Example 2 The lipid nanoparticle delivery efficiency was ≥90%; the inhibition rates in SH-SY5Y cells and SK-N-MC cells were 55.2% and 52.6%, respectively ( Figure 5-Figure 6 ).

[0215] Comparative Example 1

[0216] The difference between the lipid nanoparticles in Comparative Example 1 and Example 1 is:

[0217] (1) Lipid Nanoparticles

[0218] Concentration lipid composition (4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester MC3: ((4-hydroxybutyl) azadialkyl) bis(hexane-6,1-diyl) bis(2-hexyldecanoate): 1,2-distearoylphosphatidylcholine: PEGylated lipid: cholesterol, molar ratio of 40:15:10:5:30), the delivery efficiency of the lipid nanoparticles of comparative example 1 is ≥80%; the inhibition rates in SH-SY5Y cells and SK-N-MC cells are 45.67% and 42.4%, respectively ( Figure 7-Figure 8 ).

[0219] Comparative Example 2

[0220] The difference between the lipid nanoparticles in Comparative Example 2 and Example 1 is that the ((4-hydroxybutyl) azadialkyl) bis(hexane-6,1-diyl) bis(2-hexyldecanoate) in Table 2 is replaced with SM-102 (molecular weight 710.17) of equal concentration. The rest is the same as Example 1.

[0221] Results: The delivery efficiency of lipid nanoparticles in comparative example 2 was ≥75%; the inhibition rates in SH-SY5Y cells and SK-N-MC cells were 37.19% and 36.81%, respectively ( Figure 9-10 ).

[0222] Comparative Example 3

[0223] The difference between the lipid nanoparticles in Comparative Example 3 and Example 1 is that the 4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester mc3 in Table 2 is replaced with ((4-hydroxybutyl) azadialkyl) bis(hexane-6,1-diyl) bis(2-hexyldecanoate) at an equal concentration. The rest is the same as Example 1.

[0224] Results: The delivery efficiency of lipid nanoparticles in comparative example 3 was ≥85%; the inhibition rates in SH-SY5Y cells and SK-N-MC cells were 50.54% and 52.28%, respectively ( Figure 11-Figure 12 ).

[0225] Comparative Example 4

[0226] The difference between the lipid nanoparticles in Comparative Example 3 and Example 1 is that the PEGylated lipid in Table 2 is replaced with a lipid nanoparticle with a molecular weight of 3400. The rest is the same as Example 1.

[0227] Results: The delivery efficiency of lipid nanoparticles in comparative example 4 was ≥80%; the inhibition rates in SH-SY5Y cells and SK-N-MC cells were 23.6% and 19.44%, respectively ( Figure 13-14 ).

[0228] In summary, at the cellular level, the material of Example 1 has good dispersibility, high safety, and better delivery efficiency and inhibition efficiency than the other materials.

Claims

1. A siRNA delivery system, characterized in that: The siRNA delivery system is a lipid nanoparticle; the lipid nanoparticle comprises: 4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester mc3, ((4-hydroxybutyl) azadialkyl) bis(hexane-6,1-diyl) bis(2-hexyldecanoate), 1,2-distearoylphosphatidylcholine, PEGylated lipid and cholesterol; the molecular weight of the PEGylated lipid is 2509.2; In the lipid nanoparticles, the molar ratios of 4-(N,N-dimethylamino)butyric acid (dialene) methyl ester mc3, ((4-hydroxybutyl) azadialkyl) bis(hexane-6,1-diyl) bis(2-hexyldecanoate), 1,2-distearoylphosphatidylcholine, PEGylated lipids and cholesterol are (35-40): (10-15): (10-15): (1-3): (35-40).

2. The siRNA delivery system according to claim 1, characterized in that The molecular weights of the 4-(N,N-dimethylamino)butyric acid (dialene) methyl ester mc3, ((4-hydroxybutyl) azadialkyl) bis(hexane-6,1-diyl) bis(2-hexyldecanoate), 1,2-distearoylphosphatidylcholine and cholesterol are 642.09, 766.27, 790.15 and 386.66 respectively.

3. The siRNA delivery system according to claim 2, characterized in that The lipid nanoparticles also include siRNA.

4. The method for preparing the siRNA delivery system according to any one of claims 1 to 3, characterized in that: The following steps are involved: S1. preparing lipid-ethanol solution: dissolving 4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester mc3, ((4-hydroxybutyl) azadialkyl) bis(hexane-6,1-diyl) bis(2-hexyldecanoate), 1,2-distearoylphosphatidylcholine, PEGylated lipid and cholesterol in ethanol to prepare lipid-ethanol solution; S2. Prepare nucleic acid-buffer solution: dissolve siRNA in acidic buffer; S3, mixing and collecting: the lipid-ethanol solution of step S1 and the siRNA solution of step S2 are fully mixed to form lipid nanoparticles, and the lipid nanoparticles flowing out are collected.

5. The preparation method according to claim 4, characterized in that: The acidic buffer in step S2 is Tris-HCl buffer.

6. The preparation method according to claim 4, characterized in that: The volume concentration of the lipid-ethanol solution in step S3 is 15-30%.

7. The preparation method according to claim 4, characterized in that: The concentration of siRNA in step S3 is 300-350ug / ml.

8. The preparation method according to claim 4, characterized in that: The mixing in step S3 adopts microfluidic mixing technology.

9. The preparation method according to claim 4, characterized in that: The particle size of the lipid nanoparticles in step S3 is 80-90 mm.

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