Lipid nanoparticle compound for efficiently delivering nucleic acid to immune cells

Abstract

The invention provides a lipid nanoparticle (LNP) compound for efficiently delivering nucleic acid to immune cells, and belongs to a biological medicine technology. According to the present invention, on the basis of the previously researched and developed ionizable cationic lipid 1-126, the Box-Behnken design is adopted to design 13 LNP formulas, and the microfluidic technology is adopted to complete the preparation of the LNP; by detecting the efficiency of LNP transfecting different immune cells and optimizing LNP components, the lipid nanoparticle compound for efficiently delivering mRNA in various immune cells is screened, the compound comprises 40% of cationic lipid 1-126, 16% of DSPC, 1.5% of DMG-PEG2000 and 42.5% of beta-sitosterol, and an efficient delivery system is provided for the fields of vaccine development and gene therapy.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a lipid nanoparticle complex for efficiently delivering nucleic acids to immune cells. Technical Background

[0002] Nucleic acid drugs, including mRNA, have attracted attention for their potential in treating genetic diseases, cancer, and viral infections. Nucleic acid drug delivery systems are key to achieving their therapeutic effects, among which lipid nanoparticles (LNPs) have been widely used in mRNA vaccines as an effective non-viral carrier. LNPs are composed of ionizable lipids, cholesterol, auxiliary lipids, and PEGylated lipids. By optimizing the ratio and structure of these components, the encapsulation efficiency, stability, and cellular uptake efficiency of mRNA can be improved. Although LNPs perform well in mRNA vaccines, their efficiency is insufficient in specific applications, such as targeted delivery of immune cells, resulting in limited therapeutic effects. Summary of the invention

[0003] Based on the above problems, the present invention aims to develop an LNP formula that can efficiently transfect a variety of immune cells.

[0004] First, the present invention is based on the previously developed ionizable cationic lipid 1-126, and uses the Design of Experiments (DOE) method to design the formula of lipid nanoparticles (LNP), wherein the Box-Behnken design is a commonly used response surface method in DOE, which is particularly suitable for the optimization of continuous variables. The present invention uses the Box-Behnken design method to design 13 kinds of lipid nanoparticles (LNP) formulas to explore the effects of different lipid compositions on mRNA transfection efficiency. The DOE method is a systematic experimental planning and analysis tool that can effectively reduce the number of experiments and provide detailed information on how multiple factors affect the experimental results. The combination of DOE and Box-Behnken allows researchers to quickly identify the best LNP formula without conducting a large number of experiments.

[0005] Next, the present invention uses microfluidic technology to prepare LNP according to the above 13 formulas. Microfluidic technology mixes lipids and mRNA in tiny channels to form uniform nanoparticles. This method provides good control and repeatability, ensuring the consistency and quality of LNP. After preparation, these LNPs were incubated with Hela cells respectively, and the transfection efficiency of each LNP was evaluated by measuring the expression level of mRNA. After preliminary screening, three preferred LNP formulas with high transfection efficiency were determined, namely 126A-8, 126A-10 and 126A-11, and these formulas were optimized and verified in the next stage.

[0006] In order to verify the effectiveness of the new formula, the present invention adopts a multi-cell line transfection strategy, that is, the LNP prepared according to the above preferred formula is used to transfect a variety of immune cells to further detect the efficiency of the LNP transfection of different immune cells. The target cells for transfection include Jurkat cells (a T cell leukemia cell line), THP-1 cells (a human monocytic leukemia cell line), DC2.4 cells (a mouse bone marrow-derived dendritic cell line), and RAW264.7 cells (a mouse mononuclear macrophage leukemia cell line). This cross-cell line experimental design is conducive to the comprehensive evaluation of the transfection efficiency and functional performance of the new formula in different immune cell types. The results showed that LNPs prepared from the 126A-8 formula were more suitable for transfecting T cells, monocytes, and dendritic cells; 126A-10 and 126A-11 were more suitable for dendritic cells and mononuclear macrophages. This may be because both dendritic cells DC2.4 and mononuclear macrophages RAW264.7 have strong phagocytosis or macropinocytosis, which makes it easier for them to take in larger nanoparticles 126A-10 and 126A-11, resulting in higher delivery efficiency for the latter two. In summary, the 126A-8 formula has a wider range of applications.

[0007] Furthermore, the present invention also optimizes the composition of LNP, that is, replaces the traditional cholesterol with β-sitosterol, because β-sitosterol may improve the cell uptake and lysosomal escape of LNP, thereby improving the transfection efficiency of mRNA; and accurately evaluates its potential application value in immune cells by comparing the transfection effect of the new formula with the existing commercial formula (such as SM102). The experimental results show that although the efficiency of LNP transfection Jurkat and RAW264.7 cells prepared by the formula (such as 126A-1 and 126A-8) without optimizing the composition is lower than that of commercial SM102, the replacement of cholesterol with β-sitosterol significantly increases the transfection of LNP provided by the present invention, which is 0.5 to 5 times higher than that of SM102; when transfecting THP-1 and DC2.4 cells, the addition of β-sitosterol also significantly increases the transfection efficiency of LNP. In summary, no matter which immune cells are transfected, β-sitosterol can greatly increase the transfection efficiency of LNP; and the 126A-8-β formula can efficiently transfect multiple immune cells at the same time. Its performance exceeds the currently commercially available SM102 formula and has great application prospects and commercial value.

[0008] On the one hand, the present invention provides a lipid nanoparticle complex for efficiently delivering nucleic acids to cells, wherein the lipid nanoparticle complex comprises an ionizable cationic lipid represented by formula (1) and a sterol; the sterol is one or more of cholesterol and β-sitosterol.

[0009]

[0010] Furthermore, the molar percentage of the ionizable cationic lipid to the total lipids in the lipid nanoparticle complex is 30% to 80%.

[0011] Furthermore, the molar percentage of the sterol in the total lipids of the lipid nanoparticle complex is 5% to 60%.

[0012] Furthermore, the lipid nanoparticle complex also contains a surfactant and a neutral lipid; the surfactant is at least one of DMG-PEG, DSG-PEG, PEG-DSPE, and DOPE-PEG; the neutral lipid is at least one of DOPE, DSPC, DMPC, POPC, DOPC, DPPC, DSPE, DLPC, DUPC, DOPG, 1,2-di-O-octadecenyl-5-glycero-3-phosphocholine, 1-hexadecyl-sn-glycero-3-phosphocholine, and 1-oleoyl-2-cholesterol hemisuccinyl-5-glycero-3-phosphocholine.

[0013] In some specific embodiments, the surfactant is DMG-PEG2000, and the neutral lipid is DSPC.

[0014] Furthermore, the molar percentage of the surfactant in the total lipids of the lipid nanoparticle complex is 0.5%-5%; the molar percentage of the neutral lipid in the total lipids of the lipid nanoparticle complex is 3%-35%.

[0015] Furthermore, the cells include at least one of dendritic cells, monocytes, granulocytes, mast cells, T cells, B cells, K cells and NK cells.

[0016] In some specific embodiments, the molar percentage of the ionizable cationic lipids in the total lipids is 40%-60%, the molar percentage of the neutral lipids in the total lipids is 4%-20%, the molar percentage of the surfactants in the total lipids is 0.5%-2.5%, and the molar percentage of the sterols in the total lipids is 17.5%-55.5%.

[0017] Furthermore, the neutral lipid is DSPC, and the surfactant is DMG-PEG2000.

[0018] When the immune cells are any one or more of T cells, monocytes, dendritic cells and mononuclear macrophages, the molar percentage of the ionizable cationic lipids in the total lipids is 40%, the molar percentage of the neutral lipids in the total lipids is 16%, the molar percentage of the surfactant in the total lipids is 1.5%, and the molar percentage of the sterols in the total lipids is 42.5%. The formula is 126A-8 or 126A-8-β mentioned in the present invention, and the sterol is preferably β-sitosterol (126A-8-β); or the molar percentage of the ionizable cationic lipids in the total lipids is 50%, the molar percentage of the neutral lipids in the total lipids is 10%, the molar percentage of the surfactant in the total lipids is 1.5%, and the molar percentage of the sterols in the total lipids is 38.5%, and the sterol is cholesterol or β-sitosterol. The formula is 126A-1 or 126A-1-β mentioned in the present invention, preferably β-sitosterol (126A-1-β).

[0019] When the immune cells are any one or more of dendritic cells and mononuclear macrophages, the molar percentage of the ionizable cationic lipids in the total lipids is 40%, the molar percentage of the neutral lipids in the total lipids is 10%, the molar percentage of the surfactant in the total lipids is 0.5%, and the molar percentage of the sterols in the total lipids is 49.5%. The sterols are cholesterol or β-sitosterol, preferably cholesterol. The preferred formula is 126A-10 in the present invention; or the molar percentage of the ionizable cationic lipids in the total lipids is 50%, the molar percentage of the neutral lipids in the total lipids is 4%, the molar percentage of the surfactant in the total lipids is 0.5%, and the molar percentage of the sterols in the total lipids is 45.5%. The sterols are cholesterol or β-sitosterol, preferably cholesterol. The preferred formula is 126A-11 in the present invention.

[0020] On the other hand, the present invention provides a use of β-sitosterol for preparing a preparation for improving the efficiency of lipid nanoparticle transfection into cells, wherein the lipid nanoparticle comprises an ionizable cationic lipid represented by formula (1) and β-sitosterol.

[0021] When other components and their contents of LNP remain unchanged, replacing cholesterol with β-sitosterol can greatly improve the transfection efficiency of LNP, up to 3 times.

[0022] Further, the molar percentage of the ionizable cationic lipid in the total lipids is 50%, the molar percentage of β-sitosterol in the total lipids is 38.5%, and in some more specific embodiments, the lipid nanoparticles further comprise 10% DSPC and 1.5% DMG-PEG2000, i.e., the 126A-1-β formula mentioned in the present invention;

[0023] Or the molar percentage of the ionizable cationic lipids in the total lipids is 40%, and the molar percentage of β-sitosterol in the total lipids is 42.5%. In some more specific embodiments, the lipid nanoparticles further contain 16% DSPC and 1.5% DMG-PEG2000, which is the 126A-8-β formula mentioned in the present invention.

[0024] More preferably, 126A-8-β formulation.

[0025] On the other hand, the present invention provides a use of a lipid nanoparticle complex for preparing an agent for improving the efficiency of transfection of T cells and / or monocytes and / or dendritic cells and / or mononuclear macrophages, wherein the lipid nanoparticle complex comprises the following components: 40% of an ionizable cationic lipid represented by formula (1), 16% DSPC, 1.5% DMG-PEG2000 and 42.5% of a sterol, wherein the sterol is any one of cholesterol and β-sitosterol, i.e., the 126A-8 or 126A-8-β formula mentioned in the present invention. In some specific embodiments, the sterol is β-sitosterol, i.e., the 126A-8-β formula mentioned in the present invention; or 50% of the ionizable cationic lipid represented by formula (1), 10% DSPC, 1.5% DMG-PEG2000 and 38.5% of the sterol, wherein the sterol is one or more of cholesterol and β-sitosterol, i.e., the 126A-1 or 126A-1-β formula mentioned in the present invention. In some specific embodiments, the sterol is β-sitosterol, i.e., the 126A-1-β formula mentioned in the present invention.

[0026] On the other hand, the present invention provides a use of a lipid nanoparticle complex for preparing an agent for improving the efficiency of transfection of dendritic cells and / or mononuclear macrophages, wherein the lipid nanoparticle complex comprises the following components: 40% of the ionizable cationic lipid represented by formula (1), 10% DSPC, 0.5% DMG-PEG2000 and 49.5% cholesterol, i.e., the 126A-10 formula mentioned in the present invention; or 50% of the ionizable cationic lipid represented by formula (1), 4% DSPC, 0.5% DMG-PEG2000 and 45.5% cholesterol, i.e., the 126A-11 formula mentioned in the present invention.

[0027] The beneficial effects of the present invention include:

[0028] 1. The present invention is based on DOE, and after several rounds of screening, a new formula combination with the best effect is finally obtained, namely 126A-8-β, which is specially designed for efficient nucleic acid delivery to immune cells; in addition, by precisely adjusting the lipid components and their ratios, cholesterol is replaced with β-sitosterol, which optimizes the interaction between LNP and immune cells, thereby significantly improving the cell transfection efficiency of mRNA;

[0029] 2. The preferred new formula 126A-8-β of the present invention exhibits high transfection efficiency on a variety of immune cells;

[0030] 3. The LNP formula provided by the present invention is easy to produce on a large scale, has a simple preparation process, and is cost-controllable, suitable for industrial production, and is conducive to the commercial application of mRNA drugs;

[0031] 4. Since the LNP prepared by 126A-8-β can efficiently transfect cells, a smaller mRNA dose can achieve the same effect during the treatment process, which reduces the cost of treatment, can reduce resource consumption, and has economic benefits.

[0032] In summary, the novel lipid nanoparticles provided by the present invention have the advantages of efficient transfection of immune cells, large-scale production potential, and huge economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0034] Figure 1 :The effect of LNP particles transfecting Hela cells;

[0035] Figure 2 : Effect of LNP particle transfection on Jurkat cells; Figure A is the mean fluorescence intensity, and Figure B is the result of normalizing Figure A with 126A-1 as the baseline; MFI: mean fluorescence intensity of a single cell (flow cytometry);

[0036] Figure 3 : The effect of LNP particles transfecting THP-1 cells; Figure A is the mean fluorescence intensity, and Figure B is the result of normalizing Figure A with 126A-1 as the baseline; MFI: mean fluorescence intensity of a single cell (flow cytometry);

[0037] Figure 4 : Effect of LNP particle transfection on DC2.4 cells; Figure A is the mean fluorescence intensity, and Figure B is the result of normalizing Figure A with 126A-1 as the baseline; MFI: mean fluorescence intensity of a single cell (flow cytometry);

[0038] Figure 5: Effect of LNP particle transfection on RAW264.7 cells; Figure A is the mean fluorescence intensity, and Figure B is the result of normalization of Figure A with 126A-1 as the baseline; MFI: mean fluorescence intensity of a single cell (flow cytometry);

[0039] Figure 6 : The effect of LNP particles containing β-sitosterol on transfection of Jurkat cells; Figure A is the result of flow cytometry sorting, and Figure B is the result of quantification and homogenization of Figure A with SM102 particles as control; -β represents that the sterol in the LNP is β-sitosterol;

[0040] Figure 7 : The effect of transfecting THP-1 cells with LNP particles containing β-sitosterol; Figure A is the result of flow cytometry sorting, and Figure B is the result of quantification and homogenization of Figure A using SM102 particles as a control; -β represents that the sterol in the LNP is β-sitosterol;

[0041] Figure 8 : Effect of transfection of DC2.4 cells with LNP particles containing β-sitosterol; Figure A is the result of flow cytometry sorting, and Figure B is the result of quantification and homogenization of Figure A with SM102 particles as control; -β represents that the sterol in the LNP is β-sitosterol;

[0042] Fig. 9 : The effect of LNP particles containing β-sitosterol on transfection of RAW264.7 cells; Figure A is the flow cytometry sorting result, and Figure B is the quantification and homogenization result of Figure A using SM102 particles as a control; -β represents that the sterol in the LNP is β-sitosterol. DETAILED DESCRIPTION

[0043] The present invention is further elaborated in detail below in conjunction with the accompanying drawings and specific embodiments of the specification. The embodiments are only used to explain the present invention and are not used to limit the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work are within the scope of protection of the present invention.

[0044] It should be noted that An (n=1-13) and 126A-n (n=1-13) described in the present invention are the same formula / nanoparticle, such as A-1 and 126A-1 are the same formula / nanoparticle, but they are named differently according to the inventor's own habits; 126 refers to the new cationic lipid 1-126.

[0045] The sources of the instruments and reagents used in the present invention are as follows: Microfluidic device (INano TML / L+ fully automatic nano-drug preparation system, Maiana); microfluidic chip (Miana, R-SHM); sodium citrate buffer (McLean, S924872); dialysis bag (biotechnology grade dialysis bag 20000D RC membrane dry type, Spectrum (133342) or source leaf (SP133342)); microscope (Olympus inverted biological microscope, CKX53); flow cytometer (Attune TM NxT flow cytometer, Thermo Fisher, A24858);

[0046] The novel cationic lipid 1-126 was prepared according to the preparation method of cationic lipid 5 described in patent CN 117024323 A; DSPC (Aiweituo); DMG-PEG 2000 (Aiweituo); cholesterol (Aiweituo); β-sitosterol (AMEKO); Luciferase mRNA (EZ Cap TM Firefly Luciferase mRNA (5-moUTP), APEXBIO13); EGFP mRNA (EZCap TM EGFP mRNA (5-moUTP), APEXBIO, R1016);

[0047] All cells used in the present invention (such as Hela cells, Jurkat cells, THP-1 cells, DC2.4 cells and RAW264.7 cells) are derived from the cell bank of the Chinese Academy of Sciences.

[0048] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the other materials, reagents, etc. used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0049] Example 1: Design of LNP formulation using experimental design method

[0050] In order to screen out the LNP formula with the best transfection effect, we adopted the DOE method and used the Box-Behnken design to set the proportion of ionizable cationic lipids to 40%-60%, DSPC to 4%-20%, DMG-PEG2000 to 0.5%-2.5%, and cholesterol as the fourth component, which was added to make the total lipid proportion 100%. The ionizable cationic lipid is a new cationic lipid 1-126, which has been disclosed in patent CN117024323 A (lipid 1-126 described in the present invention corresponds to cationic lipid 5 in the patent), and its structural formula (Formula 1) is as follows:

[0051]

[0052] Thirteen LNP formulations with different ratios were designed using the DOE method and named A-1 to A-13, respectively. The ratios are shown in Table 1. It should be noted that the ratios of the components in A-1 described in the present invention are consistent with those of commercial SM102 nanoparticles. The difference is that the ionizable lipid in A-1 is 1-126 lipid, so A-1 is selected as the control of the present invention.

[0053] Table 1 Formulations of 13 LNPs

[0054]

[0055] Example 2: Preliminary screening of LNP formulations

[0056] 2.1 Preparation of LNPs

[0057] According to the 13 LNP formulations designed by the DOE method (Table 1), LNPs were prepared in the following steps:

[0058] (1) Microfluidic mixing: Prepare an ionizable cationic lipid-ethanol solution according to the LNP ratio; prepare Luciferase-mRNA (1 μg / μL) and dissolve it in a citric acid buffer (pH = 4.5); use the Myana microfluidic device to quickly mix the prepared lipid-ethanol solution and the mRNA solution in a micromixer to form LNP, with a total flow rate of 20 mL / min;

[0059] (2) Dialysis: The LNP solution was placed in a 20 KDa dialysis bag and dialyzed on a shaker at 80 rpm for 2 h. The dialysate was DPBS buffer (pH = 7.4).

[0060] (3) Ultrafiltration: Before using the 100KDa ultrafiltration tube, rinse it with pure water for 3 to 5 times; then add DPBS buffer for centrifugation and rinsing, set the centrifugal force to 13,000 rpm, and the centrifugation time to 3-5 minutes; add the sample solution to the ultrafiltration tube rinsed with DPBS, set the centrifugal force to 4,000 rpm, and the centrifugation time to 20 minutes, and collect the sample in the ultrafiltration tube;

[0061] (4) Quality control: The LNP solution was diluted to 0.5 μg / mL with DPBS buffer and 10 mM NaCl, respectively. The particle size, particle size distribution and surface charge of the LNP were characterized by dynamic light scattering (DLS) technology. The results are shown in Table 2.

[0062] Most cells have a preference for particles with medium particle size (about 50-200nm) when taking up nanoparticles, that is, LNPs in this size range can more easily bind to cell surface receptors and then be taken up by cells through endocytosis. Therefore, the particle size within this range (about 50-200nm) can enhance the affinity of LNPs to target cells, increase the accumulation of LNPs at the target location, and help improve the efficiency of LNP targeted delivery. When the particle size of LNPs is too large (such as greater than 300nm), the ability of cells to take up LNPs is significantly reduced, that is, it is difficult to internalize LNPs through cell membranes or receptors, especially certain specific cells (such as T cells or other immune cells). Therefore, too large a particle size of LNPs will lead to reduced transfection efficiency, which in turn affects the delivery effect. In addition, too large or too small a particle size may also cause mechanical damage to the cell membrane, leading to increased cytotoxicity, that is, larger LNPs may cause excessive stress responses when disintegrating in cells, while too small LNPs may activate the cell's endocytosis mechanism, and may also increase solubility and release excessive loads, increasing toxicity. In summary, the effect of particle size on LNP performance is a double-edged sword: moderate particle size (50-200nm) can usually improve cell uptake efficiency, targeting, stability and sustained release effect, which helps to optimize the drug delivery or gene transfection performance of LNP; however, too large or too small particle size may bring some negative effects, such as reducing cell uptake, improving immune clearance, enhancing cytotoxicity, etc. Therefore, in the design of LNP, it is necessary to accurately control the particle size of LNP according to the specific application target and in vivo environment to achieve the best therapeutic effect. The 13 kinds of nanoparticles provided in this embodiment have moderate particle sizes (Table 2), ranging from less than 100nm to nearly 185nm, the smallest of which is A-8 and the largest is A-11, which guarantees the efficiency of nucleic acid delivery to a certain extent; in addition, it also shows that LNPs prepared according to different LNP formulas have significant differences in particle size.

[0063] The smaller the PDI, the more uniform the distribution of the sample. The PDI of the vast majority of samples provided in this embodiment is lower than 0.1, indicating that the distribution of the LNP preparation is narrower and has good uniformity. In general, this group of LNPs has certain differences in particle size and PDI, showing the distribution change of particle size during the preparation process. Most samples have good PDI and moderate Zeta potential, indicating that they have higher dispersion stability and lower aggregation tendency. LNPs with specific particle size and higher Zeta potential may show better stability and cell uptake effect in certain biological applications. In general, the higher the absolute value of Zeta potential, the stronger the electrostatic repulsion between particles, thereby preventing particle aggregation, however, when particles are in contact with cells, excessively high charge may hinder their interaction with cell membranes, making cell uptake difficult, therefore, when designing nanoparticles for cell uptake, it is necessary to strike a balance between Zeta potential, charge properties and biocompatibility.

[0064] Table 2 Characterization of LNPs in library A

[0065]

[0066] 2.2 Transfection of Hela cells

[0067] 24 hours in advance, Hela cells were seeded in a 96-well plate at a density of 10,000 cells / well; the LNPs prepared in the above steps were added to the Hela cells (the final mRNA concentration was 1 μg / mL) and incubated in an incubator at 37°C for 48 hours; then the DMEM medium (Source Culture, L110KJ) containing 10% FBS (Opcel, BS-1105-500ML*10) and 100U / mL penicillin and 100μg / mL streptomycin (Cat. No. MA0110) was removed, and DMEM medium containing 1.5mg / mL D-fluorescein sodium (Yisheng, 40901ES03) was added, and incubated in an incubator at 37°C for about 5 minutes; finally, the transfection effect of LNP on Hela was evaluated by detecting bioluminescence with an ELISA instrument. Three replicates were set for each experimental group. The results are shown in Figure 2. Figure 1 As shown. The higher the fluorescence value, the higher the corresponding LNP transfection efficiency. Figure 1 It can be seen that the LNPs prepared according to the formulations 126A-8, 126A-10, and 126A-11 respectively have better transfection effects, so these three preferred formulations were preliminarily screened out.

[0068] Example 3: Preference of LNP transfection subjects

[0069] 3.1 Preparation of LNPs

[0070] The reporter mRNA used in this example is EGFP-mRNA (1 μg / μL), and the remaining steps are the same as those described in Example 2.

[0071] 3.2 Transfection of Jurkat cells

[0072] Jurkat cells are an acute T-cell leukemia cell line that has important applications in immunology research, especially in the study of T-cell activation and its signal transduction. Jurkat cells were seeded in a 24-well plate at a density of 200,000 cells / well; the LNPs prepared in the above steps were added to the Jurkat cells (the final concentration of mRNA was 1 μg / mL), incubated in an incubator at 37°C for 48 hours, centrifuged at 1,000 rpm for 3 minutes, and the RPMI-1640 medium (Source Culture, L210KJ) containing 10% FBS (Opcel, BS-1105-500ML*10) and 100U / mL penicillin and 100μg / mL streptomycin (Cat. No. MA0110) was removed, and the cells were washed with DPBS buffer (pH=7.4) and resuspended, and the EGFP signal was detected by flow cytometry. The results are shown in Figure 2. Figure 2 As shown, in Jurkat cells, the transfection efficiency of LNPs prepared according to the 126A-8 formula was significantly increased, more than 2 times, compared with the control group (126A-1 group); therefore, when preparing LNPs for transfection of Jurkat cells, the 126A-8 formula is preferably used.

[0073] 3.3 Transfection of THP-1 cells

[0074] THP-1 cells are a human monocytic leukemia cell line. THP-1 cells have phagocytic function and can differentiate into macrophages. They are often used to study the function of the immune system and inflammatory response. THP-1 cells were seeded in a 24-well plate at a density of 200,000 cells / well; the LNPs prepared in the above steps were added to the THP-1 cells (the final mRNA concentration was 1 μg / mL), incubated in an incubator at 37°C for 48 hours, and then centrifuged at 1,000 rpm for 3 minutes, the culture medium was removed, and the cells were washed with DPBS and resuspended, and EGFP was detected by flow cytometry. The results are shown in Figure 3 As shown, compared with the control group (126A-1 group), the transfection efficiency of LNPs prepared by 126A-8 formula increased to about 1.5 times. Therefore, when preparing LNPs for transfection of THP-1 cells, it is preferred to use 126A-8 formula.

[0075] 3.4 Transfection of DC2.4 cells

[0076] DC2.4 cells are a dendritic cell line derived from mouse bone marrow. DC2.4 cells can phagocytose and present antigens and are often used in the study of antigen presentation and immune response. DC2.4 cells were seeded in a 24-well plate at a density of 200,000 cells / well; the LNPs prepared in the above steps were added to the DC2.4 cells (the final mRNA concentration was 1 μg / mL), incubated in an incubator at 37°C for 48 hours, and then centrifuged at 1,000 rpm for 3 minutes, the culture medium was removed, and the cells were washed with DPBS buffer and resuspended, and EGFP was detected by flow cytometry. The results are shown in Figure 2. Figure 4 As shown, compared with the control group (126A-1 group), the transfection efficiency of 126A-8 was slightly increased, 126A-10 increased to about 3 times, and 126A-11 increased to more than 2 times. Therefore, when preparing LNPs for transfection of DC2.4 cells, it is preferred to use 126A-8, 126A-10 and 126A-11 formulations; more preferably, 126A-10 and 126A-11 formulations.

[0077] 3.5 Transfection of RAW264.7 cells

[0078] RAW264.7 cells are a mouse mononuclear macrophage leukemia cell line. RAW264.7 cells were seeded in a 24-well plate at a density of 200,000 cells / well; the LNPs prepared in the above steps were added to the RAW264.7 cells (the final mRNA concentration was 1 μg / mL), incubated in an incubator at 37°C for 48 hours, and then centrifuged at 1,000 rpm for 3 minutes, the culture medium was removed, and then washed with DPBS buffer and resuspended, and EGFP was detected by flow cytometry. The results are shown in Figure 5 As shown, compared with the control group (126A-1 group), 126A-10 and 126A-11 increased by more than 4 times. Therefore, when preparing LNPs for transfection of RAW264.7 cells, 126A-10 and 126A-11 formulations are preferably used.

[0079] Example 4: Effect of β-sitosterol on LNP complex transfection efficiency

[0080] Previous studies have shown that β-sitosterol may promote cell uptake of LNP and reduce the probability of lysosomal degradation of LNP, thereby improving the transfection efficiency of mRNA [1-2]. Therefore, this example explores the effect of β-sitosterol on the transfection efficiency of LNP. First, LNP was prepared according to the relevant steps in Example 3. The formulation of LNP is shown in Table 3, and the characterization of the LNP is shown in Table 4; and the LNP prepared according to the formulation in Table 3 was used to transfect 4 types of immune cells. The specific steps were the same as those described in Example 3, and the transfection effect was observed with an inverted microscope; this example used commercial SM102 LNP (Avituo) as a control, and the specific results are shown in Table 4. Figure 6 to Figure 9 shown.

[0081] Table 3 LNP formulations containing β-sitosterol

[0082]

[0083] Table 4 Characterization of LNPs

[0084]

[0085] By comparing the differences in the characterization of LNPs containing cholesterol or β-sitosterol (Tables 2 and 4), it was found that β-sitosterol generally increased the particle size and PDI value of LNPs and reduced the Zeta potential. Among them, the characterization of A-1-β and A-8-β was still in a moderate range, but the particle size of A-10-β and A-11-β was larger and the PDI value was higher, which may affect its transfection efficiency.

[0086] When transfecting Jurkat cells ( Figure 6 ), although the fluorescence intensity of the 126A-1 group was slightly weaker than that of the SM102 LNP, the fluorescence intensity of the 126A-1-β group was significantly improved after replacing cholesterol with β-sitosterol. Compared with the 126A-1 group, the average fluorescence intensity increased by about 7 times; at the same time, the transfection efficiency of the 126A-8-β group was also significantly improved. This result shows that β-sitosterol can enhance the efficiency of LNP transfection of Jurkat cells, especially the combination of 126A-1 and 126A-8; and the fluorescence signal of the combination of 126A-8-β is the strongest, indicating that this formula is very suitable for preparing LNPs for transfection of Jurkat cells and has great application prospects.

[0087] When THP-1 cells were transfected ( Figure 7 ), when β-sitosterol was not present, the fluorescence intensity of the 126A-1 group was 2 to 3 times that of the control (SM102), which means that the LNP formulations provided by the present invention (126A-1 and 126A-8, Figure 3 ) has a high applicability in transfecting THP-1 cells; after replacing cholesterol with β-sitosterol, its advantage (126A-1-β group) expanded to 7-8 times, and the 126A-8-β group was more than 13 times higher than the control, which once again confirmed the role of β-sitosterol in LNP (126A-1 group and 126A-8 group) transfected cells; at the same time, the fluorescence signal intensity of the 126A-8-β group was also the highest, indicating that the 126A-8-β group is not only suitable for transfecting Jurkat cells, but also for THP-1 cells, and has a certain broad spectrum.

[0088] When DC2.4 cells were transfected ( Figure 8 ), the fluorescence intensity change trend of 126A-1 group, 126A-1-β group and 126A-8-β group was similar to that of THP-1 cells ( Figure 7 ), which indicates that 126A-1 and 126A-8 groups are highly suitable for transfection of DC2.4 cells ( Figure 4 ); on the other hand, it was confirmed again that β-sitosterol can improve the transfection efficiency of LNP (126A-1 group and 126A-8 group) and the superior performance and wide range of transfection of the 126A-8-β composition.

[0089] When transfecting RAW264.7 cells ( Fig. 9 ), the fluorescence intensity change trend of 126A-1 group, 126A-1-β group and 126A-8-β group was similar to that of Jurkat cells ( Figure 6 ), indicating that lipid replacement can also increase the efficiency of LNP transfection in RAW264.7 cells.

[0090] However, the replacement of β-sitosterol did not improve the transfection efficiency of 126A-10 and 126A-11. Combined with the characterization results (Table 4), the particle size of 126A-10-β and 126A-11-β was larger, and the PDI was also higher than 0.2, which may be that the particles themselves were unstable, and the large particle size was not easy to be taken up by cells. In general, the transfection efficiency of A-1-β and A-8-β was higher, which may be due to the appropriate combination of particle size and Zeta potential, which contributed to the stability of LNP and the efficiency of cellular endocytosis; in contrast, the larger particle size and PDI of A-10-β and A-11-β led to their poor transfection efficiency, indicating that the replacement of β-sitosterol may play a positive role in some LNPs, but its effect may be limited when the particle size is too large.

[0091] In summary, regardless of the type of immune cells to be transfected, β-sitosterol can improve the transfection efficiency of LNP complexes with moderate particle size (80-120 nm); and the LNP prepared according to the 126A-8-β formula can transfect a variety of immune cells with extremely high efficiency, and its efficiency is much higher than that of commercial LNP (SM102), indicating that the 126A-8-β composition has great application value and commercial value.

[0092] Example 5: Effect of β-sitosterol on the transfection efficiency of LNPs composed of different ionizable cationic lipids

[0093] In order to explore the synergistic effect of β-sitosterol and different ionizable cationic lipids, LNPs containing β-sitosterol and different ionizable cationic lipids were prepared in this example. First, LNPs were prepared according to the relevant steps in Example 3.

[0094] The formulas of 126A-1 / 8 and 126A-1 / 8-β were the same as those described in Example 1 (Table 1) and Example 4 (Table 3), respectively. The formula of MC3 / MC3-β was DLin-MC3-DMA (Sigma): DSPC: cholesterol / β-sitosterol: DMG-PEG2000=50:10:38.5:1.5. The formula of ALC-0315 / ALC-0315-β was ALC-0315 (Tansht ech): DSPC: cholesterol / β-sitosterol: ALC-0159 (Tanshtech) = 46.3:9.4:42.7:1.6. It should be noted that the ratio of each component in 126A-1 / 126A-1-β is consistent with that in MC3 / MC3-β. The above-mentioned LNPs were used to transfect 4 types of immune cells, and the specific steps were the same as those described in Example 3. In this example, 126A-1 and 126A-1-β were used as controls. The specific results are shown in Table 5.

[0095] Table 5 Synergistic effect of β-sitosterol combined with different ionizable cationic lipids

[0096]

[0097] As can be seen from Table 5, the type of ionizable cationic lipids will affect the transfection efficiency of LNP. In general, the order of transfection efficiency from high to low is: 126>DLin-MC3-DMA>ALC-0315; on the other hand, β-sitosterol can increase the transfection efficiency of LNPs prepared from different ionizable lipids to varying degrees, further confirming the synergistic effect of β-sitosterol. Among them, the LNPs prepared by combining 126 ionizable cationic lipids (structural formula shown in Formula 1) and β-sitosterol (126A-8-β group) have the best effect in transfecting 4 types of immune cells, and compared with cholesterol (126A-8 group), β-sitosterol (126A-8-β group) can increase the transfection rate by 26 to 129%.

[0098] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. For those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or basic features of the present invention; therefore, no matter from which point of view, the embodiments should be regarded as exemplary and non-restrictive. The scope of the present invention is limited by the attached claims rather than the above description. Therefore, it is intended to include all changes within the meaning and scope of the equivalent elements of the claims in the present invention, and any figure marks in the claims should not be regarded as limiting the claims involved.

[0099] References

[0100] [1]Patel,Siddharth et al.“Naturally-occurring cholesterol analoguesin lipid nanoparticles induce polymorphic shape and enhance intracellulardelivery of mRNA.”Nature communications vol.11,1 983.20Feb.2020,doi:10.1038 / s41467-020-14527-2

[0101] [2]Zeng,Ye et al.“Lipid nanoparticle-based mRNAcandidates elicitpotent T cell responses.”Biomaterials science vol.11,3 964-974.31Jan.2023,doi:10.1039 / d2bm01581a

Claims

1. A lipid nanoparticle complex for efficiently delivering nucleic acids to cells, characterized in that: The lipid nanoparticle complex comprises an ionizable cationic lipid represented by formula (1) and a sterol; the sterol is one or more of cholesterol and β-sitosterol.

2. The lipid nanoparticle complex according to claim 1, characterized in that The molar percentage of the ionizable cationic lipid in the total lipid of the lipid nanoparticle complex is 30% to 80%; the molar percentage of the sterol in the total lipid of the lipid nanoparticle complex is 5% to 60%.

3. The lipid nanoparticle complex according to claim 1, characterized in that The lipid nanoparticle complex further comprises a surfactant and a neutral lipid; the surfactant is at least one of DMG-PEG, DSG-PEG, PEG-DSPE, and DOPE-PEG; the neutral lipid is DOPE, DSPC, DMPC, POPC, DOPC, DPPC, DSPE, DLPC, DUPC, DOPG, 1,2-di-O-octadecenyl-5-glycero-3-phosphocholine, At least one of 1-hexadecyl-sn-glycero-3-phosphocholine and 1-oleoyl-2-cholesterol hemisuccinyl-5-glycero-3-phosphocholine.

4. The lipid nanoparticle complex according to claim 3, characterized in that The molar percentage of the surfactant in the lipid nanoparticle complex is 0.5%-5%; the molar percentage of the neutral lipid in the lipid nanoparticle complex is 3%-35%.

5. The lipid nanoparticle complex according to claim 3, characterized in that The molar percentage of the ionizable cationic lipids in the total lipids is 40%-60%, the molar percentage of the neutral lipids in the total lipids is 4%-20%, the molar percentage of the surfactants in the total lipids is 0.5%-2.5%, and the molar percentage of the sterols in the total lipids is 17.5%-55.5%.

6. The lipid nanoparticle complex according to claim 5, characterized in that The cells include at least one of dendritic cells, monocytes, granulocytes, mast cells, T cells, B cells, K cells and NK cells.

7. A use of β-sitosterol for preparing a preparation for improving the efficiency of lipid nanoparticle transfection cells, characterized in that: The lipid nanoparticles contain an ionizable cationic lipid represented by formula (1) and β-sitosterol.

8. The use according to claim 7, characterized in that The molar percentage of the ionizable cationic lipid in the total lipid is 50%, and the molar percentage of β-sitosterol in the total lipid is 38.5%; or the molar percentage of the ionizable cationic lipid in the total lipid is 40%, and the molar percentage of β-sitosterol in the total lipid is 42.5%.

9. Use of a lipid nanoparticle complex for preparing an agent for improving the efficiency of transfection of T cells and / or monocytes and / or dendritic cells and / or mononuclear macrophages, characterized in that: The lipid nanoparticle complex contains the following components: 40% of the ionizable cationic lipid shown in formula (1), 16% DSPC, 1.5% DMG-PEG2000 and 42.5% of sterol, wherein the sterol is one or more of cholesterol and β-sitosterol; or 50% of the ionizable cationic lipid shown in formula (1), 10% DSPC, 1.5% DMG-PEG2000 and 38.5% of sterol, wherein the sterol is one or more of cholesterol and β-sitosterol.

10. Use of a lipid nanoparticle complex for preparing an agent for improving the efficiency of transfection of dendritic cells and / or monocytes and macrophages, characterized in that: The lipid nanoparticle complex contains the following components: 40% of the ionizable cationic lipid represented by formula (1), 10% DSPC, 0.5% DMG-PEG2000 and 49.5% cholesterol; or 50% of the ionizable cationic lipid represented by formula (1), 4% DSPC, 0.5% DMG-PEG2000 and 45.5% cholesterol.

Images