Low-inflammation immune ionizable liposome as well as preparation method and application thereof
By modifying NAD+ with cholesterol and combining other lipid components, low-inflammatory immune ionizable liposomes are prepared, solving the problem of existing liposomes triggering high immune responses in the body, and achieving the safety and effectiveness of gene delivery.
Patent Information
- Application Number
- CN202510383031.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-28
- Publication Date
- 2025-06-27
AI Technical Summary
Existing liposomes trigger high immune responses in the body, leading to inflammation, and affecting the safety and effectiveness of gene delivery.
By modifying nicotinamide adenine dinucleotide (NAD+) with cholesterol, NAD+-Chol is prepared, and combined with other lipid components, low inflammatory immune ionizable liposomes are prepared to reduce their immunogenicity in the body.
It achieves the reduction of the immune response triggered by liposomes while delivering genes, improves the safety of liposomes and gene transfection efficiency, and reduces the increase in immune clusters within the spleen.
Smart Images
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Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of bio-nano medicine, and particularly relates to a low-inflammatory immune-ionizable liposome, a preparation method thereof, and an application thereof. Background Art
[0002] The development of mRNA / liposome nanoparticle (LNP) technology has promoted the progress of vaccines and gene therapy, and at the same time has also raised questions about immunogenicity. After the COVID-19 pandemic, liposome vaccines have been rapidly developed and clinically tested. It has been found in the tests that mRNA / LNP vaccines may induce strong humoral and cellular immunity. Therefore, it is very important to reduce the immune response caused by liposomes.
[0003] Nicotinamide adenine dinucleotide (NAD + ) is an important coenzyme, which is composed of nicotinamide, adenine, and two ribose molecules. It exists in different redox states in cells, including oxidized NAD + and reduced NADH. Some studies have shown that NAD + is closely related to the immune activity of cells. For example, in macrophages, NAD + is a key molecule in energy metabolism, and its expression level is closely related to macrophage polarization. Imbalance of macrophage polarization is often associated with various inflammatory diseases, which in turn affects fields such as tumors, ischemia-reperfusion injury, and immune tolerance. NAD + regulates its polarization state by affecting the energy metabolism of macrophages, thereby affecting immunogenicity. Therefore, it is of great significance to develop a liposome with low immunogenicity and stable delivery to the body using NAD + . Summary of the Invention
[0004] Aiming at the problems and deficiencies in the prior art, the present invention aims to provide a low-inflammatory immune-ionizable liposome, a preparation method thereof, and an application thereof. While delivering genes, it can effectively reduce immunogenicity and successfully deliver genes into the body to make up for the deficiency of high immune response of liposomes in the body.
[0005] In order to achieve the object of the present invention, the technical solution adopted by the present invention is as follows:
[0006] The first aspect of the present invention provides a preparation method for a low-inflammatory immune-ionizable liposome, comprising the following steps:
[0007] (1) Add cholesterol, nicotinamide adenine dinucleotide, and potassium carbonate to solvent A, stir and react at 75-85°C for 10-12 h, and filter to collect the precipitate;
[0008] (2) Add the precipitate obtained in step (1) to water. After dissolution, add sodium dithionite solution, and stir the reaction in the dark at room temperature for 3 - 4 h. Filter to collect the filtrate.
[0009] (3) Add solvent B and hydrochloric acid to the filtrate obtained in step (2), mix well, centrifuge, and collect the supernatant to obtain NAD + -Chol.
[0010] (4) Add the ionizable lipid, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, distearoyl phosphatidylcholine, and cholesterol mixture to solvent C, mix evenly to obtain a lipid solution. The lipid solution is freeze-dried to obtain a low-inflammatory immune ionizable liposome, wherein the cholesterol mixture is a mixture of NAD + -Chol and cholesterol.
[0011] Preferably, the preparation method of cholesterol in step (1) is as follows: Weigh 4.34 g of cholesterol formyl chloride and 2.18 g of 2-bromoethylamine hydrobromide hydrochloride, dissolve them in 50 mL of dichloromethane or chloroform, and add them to a 100 mL round-bottom flask. Transfer the reaction system to an ice-water bath, add 3 mL of fresh and dry triethylamine. After 30 min, remove the ice-water bath, and react at room temperature for 12 h. Then wash three times with 20 - 50 mL of saturated aqueous NaCl solution to remove the residual triethylamine. Finally, dry the organic phase with 5.0 g of anhydrous magnesium sulfate, filter to collect the filtrate, and distill to obtain the crude product. Recrystallize the crude product with absolute ethanol, wash twice with anhydrous acetone, and dry in vacuo to obtain cholesterol.
[0012] Preferably, the mass ratio of cholesterol, nicotinamide adenine dinucleotide, and potassium carbonate in step (1) is (2 - 7):(4 - 12):(5 - 10).
[0013] Preferably, the molar ratio of the ionizable lipid, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, distearoyl phosphatidylcholine, dihydronicotinamide-bound cholesterol, and cholesterol mixture in step (4) is (25 - 50):(20 - 40):(10 - 20):(1 - 5), and the total concentration of the lipid solution is 8 - 16 mM.
[0014] More preferably, the molar ratio of cholesterol and NAD + -Chol in the cholesterol mixture in step (4) is (1 - 4):(1 - 4).
[0015] Preferably, the dosage ratio of the precipitate and water in step (2) is (4 - 6) g:(50 - 200) mL, and the mass ratio of the precipitate and sodium dithionite solution is (2 - 3):(2 - 3); the sodium dithionite solution is adjusted to pH 8 - 9 with potassium carbonate powder.
[0016] Preferably, in step (2), filtration is carried out using a filter membrane with a particle size of 0.2 μm.
[0017] Preferably, the solvent A is any one of acetonitrile, dichloromethane or N,N-dimethylformamide, the solvent B is anhydrous methanol, C is anhydrous ethanol or methanol, and the dosage ratio of the precipitate to the solvent B is (4-6) g:(50-100) mL.
[0018] The second aspect of the present invention provides an ionizable liposome for low-inflammatory immunity prepared by the preparation method described in the first aspect.
[0019] The third aspect of the present invention provides an application of the low-inflammatory immunity ionizable liposome described in the second aspect in gene delivery or an application in the preparation of a gene delivery drug.
[0020] The fourth aspect of the present invention provides a method for gene delivery of a low-inflammatory immunity ionizable liposome. The ionizable liposome prepared in the first aspect of the present invention is dissolved, microfluidically mixed with a gene solution to be delivered, and the solvent C is removed by ultrafiltration to obtain a low-inflammatory immunity ionizable liposome encapsulating the gene.
[0021] Preferably, the gene solution is prepared by dissolving mRNA or siRNA in a citrate buffer solution, and the N / P of the nitrogen atom in the ionizable lipid in the lipid solution to the phosphorus atom in the gene solution is 6-8.
[0022] Preferably, a citrate buffer solution is used to dissolve the ionizable liposome.
[0023] Compared with the prior art, the present invention has the following advantages:
[0024] (1) In the present invention, the component cholesterol in the liposome synthesis is modified. The important coenzyme NAD in cell metabolism is used to modify cholesterol to obtain NAD-Chol, and NAD-Chol is used as a component to further synthesize the liposome NAD@SM102. The method for preparing this liposome is simple, with uniform size, about 100 nm in size, negatively charged, and can cause an increase in neutrophils and macrophages in immune cells. + Modify cholesterol to obtain NAD + -Chol, and use NAD + -Chol as a component to further synthesize the liposome NAD + @SM102. The method for preparing this liposome is simple, with uniform size, about 100 nm in size, negatively charged, and can cause an increase in neutrophils and macrophages in immune cells.
[0025] (2) The liposome NAD prepared in the present invention +@SM102 is of uniform size and has a good membrane structure. Its encapsulation efficiency is also very high, with high efficiency in cell transfection. After in vivo injection, when observing the immune cell subsets in the immune organ spleen, it was found that it can reduce the increase in immune level caused by liposomes, restore to the normal level, and improve the immunogenicity problem in liposome delivery.
[0026] (3) The present invention uses a mixture of NAD + -Chol and cholesterol to prepare ionizable liposomes, which can reduce the inflammatory response of ionizable liposomes while ensuring high gene encapsulation efficiency and transfection efficiency, and further improve the safety and clinical application potential of ionizable liposomes. BRIEF DESCRIPTION OF THE DRAWINGS
[0027] Figure 1 is an evaluation diagram of the gene transfection effect of liposomes prepared by combining Chol and dihydro-nicotinamide-conjugated cholesterol (NAD + -Chol) in different ratios. Figure A is a flow cytometry diagram of liposomes transfected with different ratios of Chol and NAD + -Chol; Figure B is a fluorescence transfection diagram of liposomes synthesized with the optimal ratio of Chol and NAD + -Chol.
[0028] Figure 2 is a comparison diagram of the nucleic acid amount before and after the membrane rupture of liposome NAD + @SM102;
[0029] Figure 3 is the particle size and potential diagram of liposome NAD + @SM102. Figure A is the particle size diagram of NAD + @SM102, and Figure B is the potential diagram of NAD + @SM102;
[0030] Figure 4 is the morphological feature diagram of liposome NAD + @SM102. Figure A is the transmission electron microscopy diagram of NAD+@SM102, Figure B is the cryo-electron microscopy diagram of NAD+@SM102, and Figure C is the AFM diagram of NAD+@SM102;
[0031] Figure 5 The effect diagram of liposome NAD + @SM102 on normal mouse immune cells (Mean±SD, n = 5; ****P < 0.0001, *P < 0.05, ns indicates no statistical significance). Figure A is the flow cytometry analysis diagram of NAD + @SM102 on adaptive immune cell populations B cells and T cells, and Figure B is NAD +Flow cytometry analysis of innate immune cell populations, macrophages, monocytes, and neutrophils by @SM102. Detailed implementation
[0032] To make the objectives, technical solutions, and advantages of the present invention clearer and more definite, the present invention will be further described in detail below through embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.
[0033] Example 1: Preparation of low-inflammatory immune ionizable liposomes
[0034] A method for preparing low-inflammatory immune ionizable liposomes, the specific steps are as follows:
[0035] (1) Weigh 4.34 g of cholesterol formyl chloride and 2.18 g of 2-bromoethylamine hydrobromide hydrochloride and dissolve them in 50 mL of dichloromethane, then add them to a 100 mL round-bottom flask. Immediately transfer the reaction system to an ice-water bath, add 3 mL of triethylamine. After 30 minutes, remove the ice-water bath and react at room temperature for 12 h. Then wash three times with 20 mL of saturated aqueous NaCl solution to remove the residual triethylamine. Finally, dry the organic phase with 5.0 g of anhydrous magnesium sulfate, filter to collect the filtrate, distill to obtain the crude product, recrystallize the crude product with absolute ethanol, wash twice with anhydrous acetone, and dry in vacuum to obtain 2-bromoacetyl cholesterol;
[0036] (2) Weigh 348.03 mg of 2-bromoacetyl cholesterol (Br-Chol) synthesized in step (1), 598.89 mg of nicotinamide adenine dinucleotide (NAD + ) and 276.412 mg of potassium carbonate (K2CO3), add them to 100 mL of acetonitrile, stir and react at 80 °C for 12 h, and filter to obtain the precipitate;
[0037] (3) Weigh 4.6 g of the precipitate prepared in step (2) and dissolve it in 100 mL of pure water to form a solution. Add 4.16 g of sodium dithionite solution, and adjust the sodium dithionite solution to pH 8-9 with potassium carbonate powder. Stir and react at room temperature in the dark for 4 h. Filter the product and filter it with a filter membrane with a particle size of 0.2 μm to collect the filtrate;
[0038] (4) Add 60 mL of methanol to the filtrate in step (3), and at the same time add 1 mL of 0.1 M hydrochloric acid, mix well, centrifuge at 10000 rpm for 5 min, collect the supernatant to obtain NAD + -Chol;
[0039] (5) Weigh the ionizable lipid SM102, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-PEG), distearoyl phosphatidylcholine (DSPC), and cholesterol mixture in a molar ratio of 50:38.5:10:1.5. Among them, the cholesterol mixture is a mixture of NAD + -Chol and cholesterol (Chol), and the molar ratio of cholesterol (Chol) to NAD + -Chol is 4:1. Add 2 mL of absolute ethanol to prepare a mixed solution with a concentration of 8 mM. The resulting solution is the lipid solution, which is placed at 4°C or stored at -20°C; the lipid solution is freeze-dried to obtain low-inflammation immune ionizable liposomes.
[0040] Example 2: Preparation of low-inflammation immune ionizable liposomes
[0041] A preparation method for low-inflammation immune ionizable liposomes is basically the same as that in Example 1, except that: in step (5), the molar ratio of cholesterol (Chol) to NAD + -Chol is 1:4.
[0042] Example 3: Preparation of low-inflammation immune ionizable liposomes
[0043] A preparation method for low-inflammation immune ionizable liposomes is basically the same as that in Example 1, except that: in step (2), the molar ratio of cholesterol (Chol) to NAD + -Chol is 1:2.
[0044] Example 4: Preparation of low-inflammation immune ionizable liposomes
[0045] A preparation method for low-inflammation immune ionizable liposomes is basically the same as that in Example 1, except that: in step (2), the molar ratio of cholesterol (Chol) to NAD + -Chol is 2:1.
[0046] Example 5: Preparation of low-inflammation immune ionizable liposomes
[0047] A preparation method for low-inflammation immune ionizable liposomes is basically the same as that in Example 1, except that: in step (2), the molar ratio of cholesterol (Chol) to NAD + -Chol is 1:1.
[0048] Example 6: Preparation of low-inflammation immune ionizable liposomes encapsulating genes
[0049] A method for preparing a low-inflammatory immunizable ionizable liposome loaded with genes, the specific steps are as follows:
[0050] (1) Prepare 50 mL each of 100 mM citric acid monohydrate and sodium citrate dihydrate solutions respectively. Take 33 mL of the citric acid solution and 17 mL of the sodium citrate dihydrate solution, and adjust the pH to 4 with NaOH. Then, make up the volume to 100 mL with ultrapure water, add DEPC with a final concentration of 0.1%, let it stand for 30 min, and sterilize under high pressure to remove DEPC, thus obtaining 50 mM citrate buffer solution;
[0051] (2) According to N / P = 8 of the lipid solution and the gene solution, calculate that the concentration of the gene solution is 0.054 μg / μL. Dissolve the mRNA with the citrate buffer solution to make a gene solution with a concentration of 0.054 μg / μL. Use microfluidics (FluidicLab) to dissolve the low-inflammatory immunizable ionizable liposome prepared in Example 1 with the citrate buffer solution. Take 0.7 mL of the dissolved solution and 2.1 mL of the gene solution, and fully and rapidly form LNP with uniform particle size in a mixer. Then, use the ultrafiltration method to retain the synthesized LNP, replace and remove ethanol with the citrate buffer solution, and replace the solution system with a neutral buffer solution to obtain a low-inflammatory immunizable ionizable liposome loaded with genes.
[0052] Example 7: Preparation of a low-inflammatory immunizable ionizable liposome loaded with genes
[0053] A method for preparing a low-inflammatory immunizable ionizable liposome loaded with genes is basically the same as the preparation method in Example 6, the difference is that: in step (2), the low-inflammatory immunizable ionizable liposome prepared in Example 2 is used.
[0054] Example 8: Preparation of a low-inflammatory immunizable ionizable liposome loaded with genes
[0055] A method for preparing a low-inflammatory immunizable ionizable liposome loaded with genes is basically the same as the preparation method in Example 6, the difference is that: in step (2), the low-inflammatory immunizable ionizable liposome prepared in Example 3 is used.
[0056] Example 9: Preparation of a low-inflammatory immunizable ionizable liposome loaded with genes
[0057] A method for preparing a low-inflammatory immunizable ionizable liposome loaded with genes is basically the same as the preparation method in Example 6, the difference is that: in step (2), the low-inflammatory immunizable ionizable liposome prepared in Example 4 is used.
[0058] Example 10: Preparation of a low-inflammatory immunizable ionizable liposome loaded with genes
[0059] A method for preparing a low-inflammatory immune-ionizable liposome loaded with genes is basically the same as the preparation method in Example 6, except that in step (2), the low-inflammatory immune-ionizable liposome prepared in Example 5 is used.
[0060] Example 11: Evaluation of the gene transfection effect of liposomes
[0061] The main function of liposomes is to deliver genes or small molecule drugs into cells or target organs to play their roles. Therefore, the transfection effect of liposomes is crucial. To compare the transfection effect of the liposomes of the present invention, the liposomes loaded with genes prepared in Examples 6-10 were used to transfect HEK-293T cells (in order to facilitate fluorescence detection, the mRNA in the gene solutions of Examples 6-10 was replaced with mRNA with GFP tags). Among them, the liposome loaded with genes prepared in Example 6 was used to transfect HEK-293T cells and was denoted as Chol:NAD + 4:1, the liposome loaded with genes prepared in Example 7 was used to transfect HEK-293T cells and was denoted as Chol:NAD + 1:4, the liposome loaded with genes prepared in Example 8 was used to transfect HEK-293T cells and was denoted as Chol:NAD + 1:2, the liposome loaded with genes prepared in Example 9 was used to transfect HEK-293T cells and was denoted as Chol:NAD + 2:1, the liposome loaded with genes prepared in Example 10 was used to transfect HEK-293T cells and was denoted as Chol:NAD + 1:1.
[0062] At the same time, for comparison, a negative blank control group (denoted as the Control group), a positive control group (denoted as the SM102 group), and the SM102@NAD + group were set up.
[0063] The negative blank control Control directly transfected HEK-293T cells with pure mRNA with GFP tags. The SM102 group was prepared by configuring a lipid solution with a concentration of 8 mM from SM102, DSPE-PEG, DSPC, and cholesterol at a molar ratio of 50:38.5:10:1.5. Then, this lipid solution and mRNA with GFP tags were used to prepare liposomes according to the preparation method of ionizable liposomes loaded with genes described in Example 6, and HEK-293T cells were transfected with this liposome. The SM102@NAD + group is SM102, DSPE-PEG, DSPC, and NAD +-Chol was configured into a lipid solution with a concentration of 8 mM according to a molar ratio of 50:38.5:10:1.5. This lipid solution and the mRNA with GFP tag were prepared into liposomes according to the method for preparing ionizable liposomes for encapsulating genes described in Example 6, and HEK-293T cells were transfected with this liposome.
[0064] The specific operation steps for transfecting HEK-293T cells in each group were as follows:
[0065] (1) Digestion of HEK-293T cells.
[0066] Select HEK-293T cells between P3-P15. Discard the supernatant and wash with PBS. Digest with 1 mL of trypsin at 37 °C for 1 min. Use 2 mL of DMEM containing 10% FBS to terminate digestion. Gently pipette the cells to collect the cell suspension.
[0067] (2) Cell plating
[0068] Centrifuge at 1000 rpm at room temperature for 5-10 min. Discard the supernatant and resuspend the cells with 3 mL of DMEM containing 10% FBS. Count the cells. Take 5×10 4 cells / well and plate them in a 24-well plate. Add 1 mL of DMEM medium containing 10% FBS to each well.
[0069] (3) Cell transfection
[0070] Add the experimental samples, 500 ng to each well, and fluorescence can be observed after culturing for 24 h.
[0071] (4) Detection
[0072] After 48 h of transfection, collect the cells and detect the positive rate of GFP signal by flow cytometry.
[0073] The results are as Figure 1 shown. It can be seen from Figure 1 A that when the molar ratio of Chol and NAD + -Chol is 4:1, the transfection effect is the best. It can be seen from Figure 1 B that the effect of the control group SM102@NAD+ is similar to that of the positive control group SM102, and there is a transfection effect. However, when the molar ratio of Chol and NAD + -Chol is 4:1, the expression of green fluorescent protein is significantly enhanced. Subsequently, we confirmed this result according to the fluorescence intensity (as Figure 1 shown in B), where Light is the cell morphology under the light microscope to determine the cell position; merge is to combine different fluorescence signals into one image to confirm that the cells in the fluorescence image and the cells in the light microscope are in the same field of view. We found that Chol and NAD+ When the molar ratio of Chol to -Chol is 4:1, the expression of green fluorescent protein is significantly enhanced.
[0074] Example 12: Liposome NAD + Characterization of @SM102
[0075] According to the transfection effect evaluation experiment of gene delivery by liposomes in Example 11, when the molar ratio of Chol to NAD -Chol is 4:1, the transfection effect is the best. Therefore, the liposome encapsulating the gene prepared in Example 6 is denoted as NAD + @SM102 for the characterization experiment. + @SM102 was used for the characterization experiment.
[0076] (1) Determination of encapsulation efficiency
[0077] The fluorescence nucleic acid stain Quant-iT TM RNA kit was used to detect the amount of nucleic acid free (Empty LNPs) not encapsulated by LNP in the 50 μg / mL NAD + @SM102 solution. Then, the liposome was lysed with 1% Triton-100 to release the encapsulated nucleic acid, and the total nucleic acid amount in the NAD TM @SM102 solution after lysis was detected with the Quant-iT + RNA kit. The drug loading amount was obtained by calculating the difference in nucleic acid amount before and after lysis, and then divided by the total nucleic acid amount to obtain the encapsulation efficiency (Encapsulation efficiency (%) = (Quantification after lysis - Quantification before lysis) / Quantification after lysis).
[0078] The nucleic acid amounts before and after lysis are as Figure 2 shown, and the encapsulation efficiency of the liposome was calculated to be about 90%.
[0079] (2) Physicochemical property characterization
[0080] The ionizable lipid SM102 and NAD + @SM102 were respectively dispersed and diluted in ultrapure water, and their particle size and potential were tested. The hydrated particle size and potential were detected by a nanoparticle size analyzer and a Zeta potential instrument, and the results are shown in Figure 3 .
[0081] From Figure 3 A, it can be observed that the particle size ranges of both the ionizable lipid SM102 and NAD + @SM102 are about 100 nm. Figure 3 In B, it can be observed that compared with the ionizable lipid SM102, NAD +The surface negative charge of @SM102 is lower, indicating that the aqueous dispersion of the liposomes modified by NAD + is better.
[0082] (3) Morphology characterization
[0083] In order to evaluate whether the synthesized liposomes can encapsulate mRNA, morphology characterization was carried out, and the results are shown in Figure 4 . The morphology of the liposomes encapsulating genes in Example 6 was observed by transmission electron microscopy, showing round vesicles. This result indicates that the synthesized lipid nanoparticles have a uniform morphology, and the incorporation of NAD + -Chol does not affect the morphology of the lipid vesicles (as shown in Figure 4 A).
[0084] Since liposomes have a membrane-like structure, in order to further determine the layered structure of liposomes, cryo-electron microscopy was used to observe the layered structure of liposomes. The results showed that the synthesized liposomes have a monolayer structure and are completely fused with mRNA (as shown in Figure 4 B).
[0085] Since both transmission electron microscopy and cryo-electron microscopy observe the structure of liposomes in a planar state, atomic force microscopy (AFM) was used to further understand the three-dimensional structure of liposomes. As can be observed from Figure 4 C, NAD + @SM102 is a spherical structure, which is consistent with the results of the particle size measured previously.
[0086] Example 13: Effects of liposome NAD + @SM102 on immune cells in mice
[0087] Flow cytometry was used to analyze the subpopulations of immune cells in mouse blood. After injecting different liposomes, flow cytometry was used to study the subpopulations of adaptive immune cells CD45, CD11b, Ly6G, and F4 / 80 and innate immune cells CD45, CD3e, and B220 in the blood, evaluate the effects of liposomes on in vivo immune subpopulations, understand the in vivo immune subpopulations of liposomes, and explore the immunotherapy of liposomes.
[0088] To verify the effects of liposomes on mouse immunity, mice were divided into a control group (denoted as the Control group), an SM102 group, and a NAD + @SM102 group, with 5 mice in each group. NAD +In the SM102 group, mice were intravenously injected via the tail vein with a lipid solution prepared by combining cholesterol and dihydronicotinamide at a molar ratio of cholesterol to dihydronicotinamide of 4:1 and a total concentration of 1 mg / mL to synthesize blank liposomes according to the method of Example 1; in the SM102 group, mice were intravenously injected via the tail vein with a lipid solution prepared by configuring SM102, DSPC, DSPE-PEG, and Chol at a molar ratio of 50:38.5:10:1.5 with a Chol concentration of 1 mg / mL to synthesize blank liposomes according to the method of Example 1; in the Control group, PBS was injected, and the injection dose for all three groups was 5 mg / kg.
[0089] At 24 h after injection of the liposomes, after the mice were euthanized, the spleens were removed respectively. The spleens were placed in a petri dish on ice, and the spleens were cut into small pieces with sterile ophthalmic scissors, with the size as uniform as possible, about 1-2 mm 3 . Then, the piston of a syringe or gently grinding the small spleen pieces was used to release the cells from the spleen tissue. The ground cell suspension was filtered through a 100-μm cell sieve into a new centrifuge tube to remove unground tissue blocks and cell clusters. A small amount of PBS buffer can be used to rinse the cell sieve to ensure that as many cells as possible pass through. The filtered cell suspension was centrifuged at 4 °C and 300-500 g for 5-10 minutes to precipitate the cells at the bottom of the centrifuge tube. The supernatant was discarded, and an appropriate amount of erythrocyte lysate (generally 1-2 mL of lysate was added per 107 cells) was added, and the cell precipitate was gently resuspended and incubated at room temperature for 3-5 minutes. During this period, the centrifuge tube can be gently inverted several times to fully lyse the erythrocytes. An equal volume of pre-cooled PBS buffer was added to terminate the erythrocyte lysis, and then centrifuged at 4 °C and 300-500 g for 5-10 minutes, and the supernatant was discarded. The cells were washed with PBS buffer 1-2 times repeatedly to thoroughly remove erythrocyte lysis products and other impurities. Finally, the cell precipitate was resuspended with an appropriate amount of culture medium or a suitable buffer (such as RPMI-1640 medium, etc.) to disperse the cells evenly to obtain a single-cell suspension. The control group, SM102, and MAD + @The single-cell suspensions obtained from the SM102 group were evenly divided into 8 tubes and set up as negative control tubes (only PBS was added), 6 single-positive tubes (CD45, CD3e, B220, CD11b, Ly6G, F4 / 80 were added respectively), and 1 full-staining tube (CD45, CD3e, B220, CD11b, Ly6G, F4 / 80 were all added). After incubation on ice for 30 min, flow cytometry FACS CanoⅡ (BD Biosciences, USA) was used for detection, and data were collected for subsequent analysis.
[0090] First, all CD45-labeled cells with common leukocyte antigen were circled, and then the changes of subsequent immune cells were analyzed. The results were as Figure 5 shown. Compared with the control group, SM102 and NAD+ @SM102 has no effect on the overall immune cells of mice. Subsequently, further analysis of innate immune cells and adaptive immune response cells found that the T cells labeled with CD3e and B cells labeled with B220 in the adaptive immune response cells did not change in the immune population( Figure 5 A). After finding that the adaptive immune cells were not significantly improved, the innate immune response cells were analyzed. CD11b is the alpha chain of integrin CR3 and is mainly expressed on macrophages, monocytes, neutrophils, and NK cells. It was found that the expression of CD11b increased in vivo in SM102, indicating a change in its immune response. Further analysis in the CD11b cell population found that both Ly6G-labeled neutrophils and F4 / 80-labeled macrophages in bone marrow-derived cells increased( Figure 5 B), indicating that the injection of liposome SM102 causes an increase in inflammatory immunity in vivo. However, the modified liposome NAD + @SM102 significantly improved this phenomenon and has good safety and low immunogenicity.
Claims
1. A method for preparing low-inflammatory immune ionizable liposomes, characterized in that: The following steps are involved: (1) adding cholesterol, nicotinamide adenine dinucleotide and potassium carbonate to solvent A, stirring and reacting at 75-85° C. for 10-12 h, and filtering and collecting the precipitate; (2) adding the precipitate obtained in step (1) to water, adding sodium dithionite solution after dissolving, stirring and reacting for 3 to 4 hours at room temperature in the dark, and filtering and collecting the filtrate; (3) Add solvent B and hydrochloric acid to the filtrate obtained in step (2), mix thoroughly, centrifuge, collect the supernatant, and obtain NAD + -Chol; (4) adding ionizable lipids, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, distearoylphosphatidylcholine, and a cholesterol mixture to solvent C, and mixing them evenly to obtain a lipid solution. The lipid solution is freeze-dried to obtain low-inflammatory immune ionizable liposomes, wherein the cholesterol mixture is NAD + -A mixture of Chol and cholesterol.
2. The preparation method according to claim 1, characterized in that: In the step (1), the mass ratio of cholesterol, nicotinamide adenine dinucleotide and potassium carbonate is (2-7):(4-12):(5-10).
3. The preparation method according to claim 2, characterized in that: In the step (4), the molar ratio of the ionizable lipid, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, distearoylphosphatidylcholine, and mixed cholesterol is (25-50):(20-40):(10-20):(1-5), and the total concentration of the lipid solution is 8-16 mM.
4. The preparation method according to claim 3, characterized in that: In step (4), cholesterol and NAD in the cholesterol mixture + The molar ratio of -Chol is (1~4):(1~4).
5. The preparation method according to claim 2, characterized in that: In the step (2), the dosage ratio of the precipitate to water is (4-6) g: (50-200) mL, the mass ratio of the precipitate to the sodium dithionite solution is (2-3): (2-3), and the pH of the sodium dithionite solution is 8-9.
6. The preparation method according to claim 1, characterized in that: The solvent A is any one of acetonitrile, dichloromethane or N,N-dimethylformamide, the solvent B is anhydrous methanol, the solvent C is anhydrous ethanol or methanol, and the usage ratio of the precipitate to the solvent B is (4-6) g: (50-100) mL.
7. A low-inflammatory immune ionizable liposome prepared by the preparation method according to any one of claims 1 to 6.
8. Use of the low-inflammatory immune ionizable liposome according to claim 7 in gene delivery or in the preparation of gene delivery drugs.
9. A method for low-inflammatory immune ionizable liposome gene delivery, characterized in that: The low-inflammatory immune ionizable liposome prepared in claim 1 is dissolved, mixed with the gene solution to be delivered by microfluidics, and the solvent C is removed by ultrafiltration to obtain the low-inflammatory immune ionizable liposome encapsulating the gene.
10. The method according to claim 9, characterized in that The gene solution is prepared by dissolving mRNA or siRNA in a citric acid buffer solution, and the N / P ratio between nitrogen atoms in the ionizable lipids in the lipid solution and phosphorus atoms in the gene solution is 6-8.