Nucleic acid nano-lipid carrier as well as preparation method and application thereof

Abstract

The invention provides a nucleic acid nano-lipid carrier as well as a preparation method and application thereof. The preparation method comprises the following steps: carrying out first mixing on total lipid and an organic solvent to obtain an organic phase; performing second mixing on nucleic acid and a buffer solution to obtain a water phase; respectively introducing the organic phase and the water phase into an organic phase pipeline and a water phase pipeline in a microfluidic multi-pipeline fluid mixing device, and carrying out third mixing to obtain the nucleic acid nano-lipid carrier; the microfluidic multi-pipeline fluid mixing equipment comprises a Y-shaped mixer, an arrow-shaped mixer or a chip-shaped mixer. According to the microfluidic multi-pipeline fluid mixing equipment disclosed by the invention, a specific feeding pipeline included angle exists due to different types, an organic phase and a water phase can be efficiently mixed, and the obtained nucleic acid nano-lipid carrier is relatively good in stability.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a nucleic acid nanolipid carrier, a preparation method and application thereof. Background Art

[0002] Nucleic acid drugs are a new type of treatment method that can intervene in the expression of disease-related genes from the source, providing new treatment options for human genetic diseases, rare diseases, tumors, chronic diseases, and infectious diseases, and are expected to become a new type of mainstream drug after small molecule chemical drugs and protein antibody drugs. The core value of nucleic acid drugs lies in their ability to overcome the difficulties in protein structure design and selectively inhibit related pathogenic proteins at the molecular level by inhibiting gene expression, targeting non-coding RNA involved in transcription and epigenetic regulation, changing mRNA splicing, upregulating target genes, and editing genes for undruggable protein targets to treat related diseases. In theory, they can also inhibit a wide range of pathogenic proteins, so new nucleic acid drugs have attracted market attention.

[0003] After many twists and turns over the years, the development of nucleic acid drugs has been quite slow, mainly facing three major problems: poor stability of nucleic acid molecules in the body, potential side effects, and insufficient efficiency of drug delivery systems (DDS).

[0004] Nucleic acid drugs need to enter cells to work. Because of their large molecular weight, the efficiency of cellular uptake and endosome escape is relatively low, so they need the help of a delivery system to achieve higher efficacy. Currently, common delivery schemes include: nanoliposomes, cationic polymers, GalNac glycosylation modification, etc. Lipids are one of the most studied and applied delivery systems for nucleic acid drugs, and nucleic acid drugs using lipid nanoparticle delivery systems have been approved. According to their properties and structures, they can be divided into liposomes (liposomes), lipid nanoparticles (nano lipid carriers, lipidnanoparticles), etc. Currently, most of the research is on nanolipid carriers containing ionizable lipids.

[0005] Generally speaking, the smaller the particle size of lipid nanoparticles, the better the cell permeability, but the worse the stability, while the larger the particle size of lipid nanoparticles, the worse the cell permeability, but the better the stability. In the production process of nano lipid carriers, the most important thing is the mixing ratio and mixing efficiency of lipid and aqueous solution. This key factor determines the size of nano lipid carriers and the efficiency of drug encapsulation. How to flexibly adjust the particle size of nano lipid carriers according to actual needs is of great significance to the application prospects of nano lipid carriers. Currently, the commonly used process technologies are: High-pressure Homogenization (HPH), Solvent Emulsification (SE), Fluid Mixing, Supercritical Fluid (SCF), etc. Among them, the fluid mixing method can prepare nano lipid carriers with smaller particle size, and has the advantages of good reproducibility and easy process amplification, which is the current mainstream development direction.

[0006] The principle of microfluidic preparation is to mix the organic phase and the aqueous phase in a mixing component at a controlled flow rate, thereby forming the organic phase into droplets. In order to obtain a nucleic acid nanolipid carrier with a suitable particle size, it is particularly important to optimize the structure and mixing conditions of the mixing component of the microfluidic device. Therefore, the present application intends to provide a method for preparing a nucleic acid nanolipid carrier with a suitable particle size. Summary of the invention

[0007] The main purpose of the present invention is to provide a nucleic acid nanolipid carrier, a preparation method and application thereof, so as to solve the problem of low drug loading efficiency of the nucleic acid nanolipid carrier prepared in the prior art.

[0008] In order to achieve the above-mentioned object, according to the first aspect of the present invention, a method for preparing a nucleic acid nanolipid carrier is provided, the preparation method comprising: first mixing total lipids with an organic solvent to obtain an organic phase; second mixing nucleic acid with a buffer solution to obtain an aqueous phase; passing the organic phase and the aqueous phase into an organic phase pipeline and an aqueous phase pipeline respectively for third mixing to obtain the nucleic acid nanolipid carrier; the flow rate ratio of the organic phase to the aqueous phase is 1:1-9; wherein the organic phase pipeline and the aqueous phase pipeline are respectively an organic phase pipeline and an aqueous phase pipeline in a microfluidic multi-channel fluid mixing device, and the microfluidic multi-channel fluid mixing device comprises: a Y-type mixer, an arrow-shaped mixer or a chip-type mixer; the chip-type mixer comprises two The invention discloses a microfluidic multi-channel fluid mixing device, comprising a sample inlet and a sample outlet, the two sample inlets are arranged in parallel, the two sample inlets are connected to the same sample outlet through an organic phase pipeline and an aqueous phase pipeline respectively, the organic phase pipeline and the aqueous phase pipeline are arranged continuously and crosswise, the organic phase pipeline and the aqueous phase pipeline are connected, the sample inlet, the sample outlet, the organic phase pipeline and the aqueous phase pipeline are all arranged on a flat plate; the angle between the organic phase pipeline and the aqueous phase pipeline is θ, when the microfluidic multi-channel fluid mixing device is a Y-type mixer, the angle θ is 80-160°, when the microfluidic multi-channel fluid mixing device is an arrow-type mixer, the angle θ is 200-300°, and when the microfluidic multi-channel fluid mixing device is a chip-type mixer, the angle θ is 60-120°.

[0009] Furthermore, when the microfluidic multi-channel fluid mixing device is a Y-type mixer or an arrow-shaped mixer, the total flow rate of the nucleic acid nanolipid carrier is 5-50 mL / min; preferably, when the microfluidic multi-channel fluid mixing device is a chip-type mixer, the total flow rate of the nucleic acid nanolipid carrier is 3-10 mL / min.

[0010] Further, the total lipids include one or more of ionizable phospholipids, neutral phospholipids, cholesterol or pegylated phospholipids; preferably, the ionizable phospholipids include: [(4-hydroxybutyl) azadiyl] bis(hexane-6,1-diyl) bis(2-hexyldecanoate), 8-[(2-hydroxyethyl) (6-oxo-6-decyloxyhexyl) amino] octanoic acid (heptadecan-9-yl) ester, 4-(N,N-dimethylamino) butyric acid (dilinoleyl) methyl ester, N2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane or (2,3-dioleoyl-propyl)-trimethylammonium chloride or 1,1 '-[(2-{4-[2-({2-[bis(2-hydroxydodecyl)amino]ethyl}(2-hydroxydodecyl)amino)ethyl]piperazine-1-yl}ethyl)azadialkyl]bis(dodecan-2-ol) one or more; preferably, the neutral phospholipids include: 1,2-distearoyl-sn-glyceryl-3-phosphatidylcholine, 1,2-dioleoyl-sn-glyceryl-3-phosphatidylcholine, 1,2-dipalmitoyl-sn-glyceryl-3-phosphatidylcholine, 2-oleoyl-1-palmitoyl-sn-glyceryl-3-phosphatidylcholine, 1,2-dioleoyl Preferably, the PEGylated phospholipids include: one or more of 1,2-dimyristoyl-rac-glyceryl-3-methoxypolyethylene glycol, 1,2-distearoyl-rac-glyceryl-3-methoxypolyethylene glycol, 1,2-dipalmitoyl-rac-glyceryl-3-methoxypolyethylene glycol or 1,2-distearoyl-sn-glyceryl-3-phosphatidylethanolamine-methoxypolyethylene glycol; preferably, the organic solvent includes: ethanol.

[0011] Furthermore, the total lipids include ionizable phospholipids, neutral phospholipids, cholesterol and pegylated phospholipids, and the concentration of ionizable phospholipids in the organic phase is 1-10 mM; preferably, the concentration of neutral phospholipids in the organic phase is 0.5-5 mM; preferably, the concentration of pegylated phospholipids in the organic phase is 0-2 mM; preferably, the concentration of cholesterol in the organic phase is 2-5 mM; preferably, in the total lipids, the molar fraction of ionizable phospholipids is 30-60%, the molar fraction of neutral phospholipids is 5%-35%, the molar fraction of cholesterol is 10%-40%, and the molar fraction of pegylated phospholipids is 1%-3%.

[0012] Furthermore, the buffer solution is selected from one or more of the following: citric acid buffer solution, phosphate buffer solution, amino acid buffer solution, Tris buffer solution or acetate buffer solution; preferably, the nucleic acid includes: siRNA, mRNA, circular RNA, microRNA, antisense nucleic acid or plasmid; preferably, the pH value of the buffer solution is 3~10; preferably, the concentration of the buffer solution is 5~500 mM.

[0013] Furthermore, the molar ratio of nucleic acid to total lipid is 1:200-800; preferably, the concentration of nucleic acid is 0.01-500 μg / mL; preferably, when total lipid includes ionizable phospholipid, the molar ratio of N / P element in ionizable phospholipid and nucleic acid is 2-20:1.

[0014] Furthermore, the mixed product after the third mixing is subjected to ultrafiltration treatment; preferably, the ultrafiltration treatment is performed using an ultrafiltration tube, wherein the molecular weight cutoff of the ultrafiltration tube is 10-100KDa.

[0015] In order to achieve the above object, according to a second aspect of the present invention, a nucleic acid nanolipid carrier obtained by the above method for preparing the nucleic acid nanolipid carrier is provided.

[0016] Furthermore, the average particle size of the nucleic acid nanolipid carrier is 50-300 nm; preferably, the particle distribution index PDI of the nucleic acid nanolipid carrier is 0.05-0.4.

[0017] In order to achieve the above object, according to the third aspect of the present invention, there is provided a use of a nucleic acid nanolipid carrier obtained by the above method for preparing a nucleic acid nanolipid carrier in the preparation of a nucleic acid drug.

[0018] By applying the technical solution of the present invention, the organic phase and the aqueous phase are mixed and prepared using the microfluidic multi-channel fluid mixing device of the present application, and a nucleic acid nano-lipid carrier with a suitable particle size and good drug loading rate and nucleic acid recovery rate can be obtained under the condition of a low organic phase and aqueous phase flow rate ratio. Among them, the microfluidic multi-channel fluid mixing device of the present application has a specific feed channel angle according to its different types, and can efficiently mix a low amount of organic phase and aqueous phase. The obtained nucleic acid nano-lipid carrier has good stability, can be mass-produced, meets the needs of clinical applications, and provides strong technical support for the development of nucleic acid drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The drawings constituting a part of the present application are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0020] Figure 1A schematic diagram of a Y-type mixing device of the present application is shown;

[0021] Figure 2 A schematic diagram of an arrow-shaped mixing device of the present application is shown;

[0022] Figure 3 A schematic diagram of a chip-type mixing device of the present application is shown;

[0023] Figure 4 A schematic diagram of a conventional T-type mixing device in the prior art is shown. DETAILED DESCRIPTION

[0024] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0025] As mentioned in the background technology, when using the fluid mixing method of the prior art to prepare nucleic acid nanolipid carriers, the organic phase and the aqueous phase cannot obtain a complete nucleic acid nanolipid carrier when the amount is small, and because the mixing device used in the preparation process has a low control ability over the material mixing, a product with a suitable particle size and stability cannot be obtained. The drug loading rate and nucleic acid recovery rate of the preparation method are low, the cell penetrability as a drug delivery carrier is poor, and the nucleic acid loading amount is low. Therefore, the present application intends to provide a method for preparing a nucleic acid nanolipid carrier with a high drug loading rate.

[0026] In a first typical embodiment of the present application, a method for preparing a nucleic acid nanolipid carrier is provided, the method comprising: first mixing total lipids with an organic solvent to obtain an organic phase; second mixing nucleic acid with a buffer solution to obtain an aqueous phase; passing the organic phase and the aqueous phase into an organic phase pipeline and an aqueous phase pipeline, respectively, for third mixing to obtain the nucleic acid nanolipid carrier; the flow rate ratio of the organic phase to the aqueous phase is 1:1-9, specifically 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8; wherein the organic phase pipeline and the aqueous phase pipeline are respectively the organic phase pipeline in the microfluidic multi-channel fluid mixing device; The microfluidic multi-channel fluid mixing device includes: a Y-type mixer, an arrow-shaped mixer or a chip-type mixer; the chip-type mixer includes two inlets and one outlet, the two inlets are arranged in parallel, the two inlets are connected to the same outlet through an organic phase pipeline and an aqueous phase pipeline respectively, the organic phase pipeline and the aqueous phase pipeline are continuously cross-arranged, the organic phase pipeline and the aqueous phase pipeline are connected, and the inlet, the outlet, the organic phase pipeline and the aqueous phase pipeline are all arranged on a flat plate; the angle between the organic phase pipeline and the aqueous phase pipeline is θ, and when the microfluidic multi-channel fluid mixing device is a Y-type mixer, the angle θ is 80-160° (such as Figure 1As shown in FIG. 2 ), when the microfluidic multi-channel fluid mixing device is an arrow-shaped mixer, the angle θ is 200-300° (as shown in FIG. Figure 2 As shown), when the microfluidic multi-channel fluid mixing device is a chip-type mixer, the angle θ is 60-120° (as shown Figure 3 As shown), and the angle of intersection of the organic phase pipeline and the aqueous phase pipeline is the same each time. When the microfluidic multi-channel fluid mixing device is a Y-shaped mixer or an arrow-shaped mixer, the total flow rate of the nucleic acid nanolipid carrier is 5-50 mL / min; preferably, when the microfluidic multi-channel fluid mixing device is a chip-type mixer, the total flow rate of the nucleic acid nanolipid carrier is 3-10 mL / min.

[0027] The present application controls the specific angle of the mixing device, so that the organic phase and the aqueous phase collide and mix at a specific angle, which can enhance the mixing strength of the aqueous phase and the organic phase, thereby effectively improving the mixing efficiency of the organic phase and the aqueous phase, and obtaining a product with a high drug loading rate and stability under the condition of low flow rates of the organic phase and the aqueous phase, achieving good industrial production quality under low energy consumption, and the method is applicable to the preparation of various nucleic acid drugs, such as siRNA, mRNA vaccines, etc. Among them, the total flow rate is the sum of the organic phase injection flow rate and the aqueous phase injection flow rate, and the injection flow rate is the same as the discharge flow rate.

[0028] In order to further improve the biocompatibility and targeting of the carrier for drug delivery, it is necessary to select suitable lipid types. In a preferred embodiment, the total lipids include one or more of ionizable phospholipids, neutral phospholipids, cholesterol or pegylated phospholipids; preferably, the ionizable phospholipids include: [(4-hydroxybutyl) azadiyl] bis(hexane-6,1-diyl) bis(2-hexyldecanoate) (ALC-0315), 8-[(2-hydroxyethyl) (6-oxo-6-decyloxyhexyl) amino] octanoic acid (heptadecan-9-yl) ester (SM-102), 4-(N,N-dimethylamino) butyric acid (dilinoleyl) methyl ester (DLin-MC3-DMA), N2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane or (2,3-dioleoyl-propyl)-trimethylammonium chloride (DOTAP) or 1,1 '-[(2-{4-[2-({2-[bis(2-hydroxydodecyl)amino]ethyl}(2-hydroxydodecyl)amino)ethyl]piperazin-1-yl}ethyl)azadialkyl]bis(dodecan-2-ol) (C12-200) one or more; preferably, the neutral phospholipids include: 1,2-distearoyl-sn-glyceryl-3-phosphatidylcholine (DSPC), 1,2-dioleoyl-sn-glyceryl-3-phosphatidylcholine (DOPC), 1,2-dipalmitoyl-sn-glyceryl-3-phosphatidylcholine (DPPC), 2-oleoyl-1-palmitoyl-sn-glyceryl-3-phosphatidylcholine (POPC), 1,2-dioleoyl-sn-glyceryl-3-phosphatidylethanolamine (DOPE) or 1,2- One or more of distearoyl-sn-glyceryl-3-phosphatidylethanolamine (DSPE); preferably, the PEGylated phospholipids include: one or more of 1,2-dimyristoyl-rac-glyceryl-3-methoxypolyethylene glycol, 1,2-distearoyl-rac-glyceryl-3-methoxypolyethylene glycol, 1,2-dipalmitoyl-rac-glyceryl-3-methoxypolyethylene glycol or 1,2-distearoyl-sn-glyceryl-3-phosphatidylethanolamine-methoxypolyethylene glycol; preferably, the organic solvent includes: ethanol.

[0029] In order to further control the concentration and molar ratio of various lipids within a suitable range, so as to obtain a carrier product with suitable charge and fluidity, and further improve the drug loading rate, in a preferred embodiment, the total lipids include ionizable phospholipids, neutral phospholipids, cholesterol and PEGylated phospholipids, and the concentration of ionizable phospholipids in the organic phase is 1-10 mM; preferably, the concentration of neutral phospholipids in the organic phase is 0.5-5 mM; preferably, the concentration of PEGylated phospholipids in the organic phase is 0-2 mM; preferably, the concentration of cholesterol in the organic phase is 2-5 mM; preferably, in the total lipids, the molar fraction of ionizable phospholipids is 30-60%, the molar fraction of neutral phospholipids is 5%-35%, the molar fraction of cholesterol is 10%-40%, and the molar fraction of PEGylated phospholipids is 1%-3%. Among them, the value of ionizable phospholipids has a great influence on the drug loading rate of nucleic acid nanolipid carriers.

[0030] In order to further protect the nucleic acid from the influence of the external environment, improve its stability and biological activity in the body, and further improve the drug loading rate of the preparation method, in a preferred embodiment, the buffer solution is selected from one or more of the following: citric acid buffer solution, phosphate buffer solution, amino acid buffer solution, Tris buffer solution or acetate buffer solution; preferably, the nucleic acid includes: siRNA, mRNA, circular RNA, microRNA, antisense nucleic acid or plasmid; preferably, the pH value of the buffer solution is 3-10, and the specific pH value of the buffer solution is 3, 4, 5, 6, 7, 8, 9, 10; preferably, the concentration of the buffer solution is 5-500 mM, and the specific concentration of the buffer solution is 5, 15, 20, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500 mM.

[0031] In order to further improve the loading efficiency and stability of nucleic acids and optimize the structure of the carrier, in a preferred embodiment, the molar ratio of nucleic acid to total lipid is 1:200-800, and the specific molar ratio is 1:200, 1:300, 1:400, 1:500, 1:600, 1:700 and 1:800; preferably, the concentration of nucleic acid is 0.01-500 μg / mL, and the specific concentration of nucleic acid is 0.5 μg / mL, 10 μg / mL, 25 μg / mL , 100μg / mL, 150μg / mL, 200μg / mL, 250μg / mL, 300μg / mL, 350μg / mL, 400μg / mL, 450μg / mL; To further improve the drug loading rate of the nucleic acid nanolipid carrier, preferably, when the total lipids include ionizable phospholipids, the N / P ratio of ionizable phospholipids and nucleic acids is 2-20:1, and the specific N / P ratio is 2:1, 5:1, 10:1, 15:1, and 20:1.

[0032] In order to further remove unbound lipids and organic solvents and purify the carrier, in a preferred embodiment, the mixed product after the third mixing is subjected to ultrafiltration treatment; preferably, the ultrafiltration treatment is carried out using an ultrafiltration tube, wherein the molecular weight cutoff of the ultrafiltration tube is 10-100KDa, and the specific molecular weight cutoff is 10KDa, 20KDa, 25KDa, 50KDa, 75KDa, and 100KDa.

[0033] In a second typical embodiment of the present application, a nucleic acid nanolipid carrier obtained by the preparation method of the above-mentioned nucleic acid nanolipid carrier is provided. The carrier has a high drug loading rate, good stability and biocompatibility, and is suitable for the preparation of various nucleic acid drugs, such as siRNA therapeutic agents, mRNA vaccines, etc., and can improve the targeting and therapeutic effect of the drug.

[0034] The carrier obtained by the above-mentioned preparation method can obtain a carrier with relatively stable particle size and distribution index PDI, reduce batch-to-batch differences, and is suitable for high-quality and high-consistency nucleic acid drug production to ensure the safety and effectiveness of clinical applications. In a preferred embodiment, the average particle size of the nucleic acid nanolipid carrier is 50-300nm; preferably, the distribution index PDI of the nucleic acid nanolipid carrier is 0.05-0.4, and the specific PDI is 0.05, 0.1, 0.2, 0.3, 0.4.

[0035] In the third typical embodiment of the present application, the use of the nucleic acid nanolipid carrier obtained by the preparation method of the nucleic acid nanolipid carrier described above in the preparation of nucleic acid drugs is provided. The carrier can effectively protect nucleic acids, improve their stability and biological activity in vivo, and is suitable for the development of various nucleic acid drugs, such as siRNA therapeutic agents, mRNA vaccines, etc., which can significantly improve the therapeutic effect, reduce side effects, and meet the high requirements of clinical treatment.

[0036] The present application is further described in detail below in conjunction with specific embodiments. These embodiments should not be construed as limiting the scope of protection claimed in the present application.

[0037] Performance testing experiment of nucleic acid nanolipid carrier

[0038] 1. Drug loading rate determination method

[0039] The nucleic acid nanolipid carriers prepared in each example were centrifuged at 4000×g for 10 min at 4°C using a 100 KDa ultrafiltration tube, and the lower layer solution was collected. The nucleic acid nanolipid carriers, the lower layer solution, and the standard curve sample (siRNA) obtained after centrifugation were diluted thirty times with 1% Triton X-100 PBS for demulsification, and then vortexed with 2×NA-Red at 200 / 50 (v / v). 200 μL of each group of samples was taken in an ELISA plate, and the fluorescence absorption intensity (Ex=300 nm, Em=600 nm) was detected. The standard curve was established using the fluorescence absorption intensity and content of the standard curve sample, and the drug loading rate and nucleic acid recovery rate of the nanolipid carrier samples obtained in each example were calculated. The calculation method is shown in the following formula, the same below.

[0040]

[0041]

[0042] The nucleic acid content is the total nucleic acid content of the nucleic acid nanolipid carrier obtained after centrifugation by fluorescence measurement; the nucleic acid free content is the total nucleic acid content of the lower layer solution by fluorescence measurement. The nucleic acid feed amount is the theoretical concentration in the subsequent effect evaluation, which is the theoretical nucleic acid concentration that should be obtained without considering the loss.

[0043] 2. Particle size determination method

[0044] The nucleic acid nanolipid carrier obtained after centrifugation in the above "1. Drug loading rate determination method" was diluted 20 times and measured using a Malvern particle size analyzer.

[0045] In addition, the preparation method of the nucleic acid nanolipid carrier in the present application can prepare the required nucleic acid nanolipid carrier using appropriate dosages as needed, including but not limited to the dosage of the organic phase in the following embodiments of the present application: 0.2 mL (based on each nucleic acid nanolipid carrier), the dosage of the aqueous phase: 0.8 mL (based on each nucleic acid nanolipid carrier).

[0046] Example 1 Preparation of Nucleic Acid Nanolipid Carrier

[0047] 1. Preparation of organic phase: Dissolve ionizable phospholipid DLin-MC3-DMA, neutral phospholipid DSPC, cholesterol Chol and pegylated phospholipid DMG-PEG2000 in ethanol by ultrasonication to obtain an organic phase. Among them, the molar fraction of ionizable phospholipids in total lipids is 50%, the molar fraction of neutral phospholipids is 10%, the molar fraction of cholesterol is 38.5%, and the molar fraction of pegylated phospholipids is 1.5%. The concentration of ionizable phospholipids in the obtained organic phase is 5 mM; the concentration of neutral phospholipids is 1 mM; the concentration of cholesterol is 3.85 mM; and the concentration of pegylated phospholipids is 0.15 mM.

[0048] 2. Prepare the aqueous phase: dissolve siRNA in a citric acid-disodium hydrogen phosphate buffer to obtain an aqueous phase, wherein the pH value of the citric acid-disodium hydrogen phosphate buffer is 4.0, the concentration of the citric acid-disodium hydrogen phosphate buffer is 200 mM, and the concentration of siRNA in the citric acid-disodium hydrogen phosphate buffer is 120 μg / mL.

[0049] In the organic phase and the aqueous phase prepared above, the molar ratio of siRNA to total lipids was 1:310.41; the N / P ratio of ionizable phospholipid DLin-MC3-DMA to siRNA was 6.88:1.

[0050] 3. Preparation of nucleic acid nanolipid carriers: The organic phase and aqueous phase prepared above are respectively introduced into the organic phase pipeline and aqueous phase pipeline of the microfluidic multi-channel fluid mixing device, wherein the microfluidic multi-channel fluid mixing device used is a chip-type, the angle between the organic phase pipeline and the aqueous phase pipeline in the device is 90°, and the flow rate ratio of the organic phase to the aqueous phase is 1:4. The obtained mixed product flows out from the discharge port of the mixing device, and the total flow rate of the chip-type mixing device is 5mL / min. Centrifugation is performed using an ultrafiltration tube with a molecular weight cutoff of 100KDa, and the filtration is repeated 3 times to obtain a nucleic acid nanolipid carrier.

[0051] 4. The results of the prepared nucleic acid lipid nanoparticles are shown in Table 1 below.

[0052] Table 1:

[0053]

[0054] Example 2 Preparation of Nucleic Acid Nanolipid Carriers

[0055] 1. Preparation of organic phase: Dissolve ionizable phospholipid DLin-MC3-DMA, neutral phospholipid DSPC, cholesterol Chol and pegylated phospholipid DMG-PEG2000 in ethanol by ultrasonication to obtain an organic phase. Among them, the molar fraction of ionizable phospholipids in total lipids is 50%, the molar fraction of neutral phospholipids is 10%, the molar fraction of cholesterol is 38.5%, and the molar fraction of pegylated phospholipids is 1.5%. The concentration of ionizable phospholipids in the obtained organic phase is 5 mM; the concentration of neutral phospholipids is 1 mM; the concentration of cholesterol is 3.85 mM; and the concentration of pegylated phospholipids is 0.15 mM.

[0056] 2. Prepare the aqueous phase: dissolve siRNA in a citric acid-disodium hydrogen phosphate buffer to obtain an aqueous phase, wherein the pH value of the citric acid-disodium hydrogen phosphate buffer is 4.0, the concentration of the citric acid-disodium hydrogen phosphate buffer is 200 mM, and the initial concentration of siRNA is 60 μg / mL.

[0057] In the organic phase and the aqueous phase prepared above, the molar ratio of siRNA to total lipids was 1:620.83; the N / P ratio of ionizable phospholipid DLin-MC3-DMA to siRNA was 13.75:1.

[0058] 3. Preparation of nucleic acid nanolipid carriers: The organic phase and aqueous phase prepared above are respectively introduced into the organic phase pipeline and aqueous phase pipeline of the microfluidic multi-channel fluid mixing device, wherein the microfluidic multi-channel fluid mixing device used is a chip-type, the angle between the organic phase pipeline and the aqueous phase pipeline in the device is 90°, and the flow rate ratio of the organic phase to the aqueous phase is 1:4. The obtained mixed product flows out from the discharge port of the mixing device, and the total flow rate of the chip-type mixing device is 5mL / min. Centrifugation is performed using an ultrafiltration tube with a molecular weight cutoff of 100KDa, and the filtration is repeated 3 times to obtain a nucleic acid nanolipid carrier.

[0059] 4. The results of the prepared nucleic acid lipid nanoparticles are shown in Table 2 below.

[0060] Table 2:

[0061]

[0062] Example 3 Preparation of Nucleic Acid Nanolipid Carriers

[0063] 1. Preparation of organic phase: Dissolve ionizable phospholipid DLin-MC3-DMA, neutral phospholipid DSPC, cholesterol Chol and pegylated phospholipid DMG-PEG2000 in ethanol by ultrasonication to obtain an organic phase. Among them, the molar fraction of ionizable phospholipids in total lipids is 50%, the molar fraction of neutral phospholipids is 10%, the molar fraction of cholesterol is 38.5%, and the molar fraction of pegylated phospholipids is 1.5%. The concentration of ionizable phospholipids in the obtained organic phase is 5 mM; the concentration of neutral phospholipids is 1 mM; the concentration of cholesterol is 3.85 mM; and the concentration of pegylated phospholipids is 0.15 mM.

[0064] 2. Prepare the aqueous phase: dissolve siRNA in citric acid-disodium hydrogen phosphate buffer to obtain an aqueous phase, wherein the pH value of the citric acid-disodium hydrogen phosphate buffer is 4.0, the concentration of the citric acid-disodium hydrogen phosphate buffer is 200 mM, and the initial concentration of siRNA is 192 μg / mL.

[0065] In the organic phase and the aqueous phase prepared above, the molar ratio of siRNA to total lipids was 1:155.21; the N / P ratio of ionizable phospholipid DLin-MC3-DMA to siRNA was 3.44:1.

[0066] 3. Preparation of nucleic acid nanolipid carriers: The organic phase and aqueous phase prepared above are respectively introduced into the organic phase pipeline and aqueous phase pipeline of the microfluidic multi-channel fluid mixing device, wherein the microfluidic multi-channel fluid mixing device used is a chip-type, the angle between the organic phase pipeline and the aqueous phase pipeline in the device is 90°, and the flow rate ratio of the organic phase to the aqueous phase is 1:4. The obtained mixed product flows out from the discharge port of the mixing device, and the total flow rate of the chip-type mixing device is 5mL / min. Centrifugation is performed using an ultrafiltration tube with a molecular weight cutoff of 100KDa, and the filtration is repeated 3 times to obtain a nucleic acid nanolipid carrier.

[0067] 4. The results of the prepared nucleic acid lipid nanoparticles are shown in Table 3 below.

[0068] Table 3:

[0069]

[0070] Example 4 Effect of different total flow rates on the particle size of nucleic acid nanolipid carriers

[0071] The operations of "1. preparing organic phase" and "2. preparing aqueous phase" in this embodiment are the same as those in Example 1, but the microfluidic multi-channel fluid mixing device and flow rate-related parameters used in "3. preparing nucleic acid nanolipid carrier" are different.

[0072] The mixing devices used in this embodiment include: an arrow-shaped mixing device in which the angle between the organic phase pipeline and the aqueous phase pipeline is 240° and a T-shaped mixing device in which the angle between the organic phase pipeline and the aqueous phase pipeline is 180° (such as Figure 4 The total flow rate and the flow rate ratio of the organic phase to the aqueous phase in different mixing devices are shown in Table 4, and the performance of the obtained product is also shown in Table 4.

[0073] If the sample particle size is 250nm~350nm, the total flow rate of the T-type mixer is 5 mL / min, while the total flow rate of the arrow-type mixer is 25 mL / min, and the sample distribution index is smaller and the sample quality is better. The higher the total flow rate, the higher the sample output rate. Therefore, within a certain particle size range, the arrow-type mixer is more efficient than the T-type mixer, which can greatly save time costs. In addition, under low flow rate conditions, the arrow-type mixer can produce samples with smaller particle size and more concentrated particle size dispersion than the T-type.

[0074] In general, the arrow-shaped mixer can produce products that save materials and time according to product requirements.

[0075] Table 4:

[0076]

[0077] Example 5 Effects of different buffer solution types on drug loading rate and recovery rate of nucleic acid nanolipid carriers

[0078] The operations of "1. Preparing organic phase" and "3. Preparing nucleic acid nanolipid carrier" in this example are the same as those in Example 1, but the buffer solution and nucleic acid concentration used in "2. Preparing aqueous phase" are different.

[0079] Among them, the specific steps of "2. Preparation of aqueous phase" in this embodiment are: dissolving siRNA in different buffer solutions to obtain aqueous phases. The pH value of the buffer solution is 4.0, the concentration of the buffer solution is 200 mM, and the initial concentrations of siRNA are 48 μg / mL and 96 μg / mL, respectively. The effects of different buffer solutions and different nucleic acid concentrations on drug loading and recovery are shown in Table 5. It can be seen that in the citric acid-disodium hydrogen phosphate buffer, the drug loading and recovery of the nanolipid carrier sample are both optimal. And the N / P ratio in Table 5 is the N / P ratio of ionizable phospholipids to nucleic acids.

[0080] Table 5:

[0081]

[0082] Example 6 Effects of different mixing devices on drug loading rate and recovery rate of nucleic acid nanolipid carriers

[0083] In this embodiment, "1. Preparation of organic phase" is the same as that in Embodiment 1, and in "2. Preparation of aqueous phase", except for the different nucleic acid concentration, the rest of the operations are the same as those in Embodiment 1, and in "3. Preparation of nucleic acid nanolipid carrier", except for the different microfluidic multi-channel fluid mixing device used, the rest are the same as those in Embodiment 1.

[0084] Among them, the initial concentrations of siRNA in this embodiment are 120 μg / mL and 240 μg / mL, respectively, and the mixing device is as shown in Table 6, including: a chip-type mixing device with an angle of 90° between the organic phase pipeline and the aqueous phase pipeline, a Y-type mixing device with an angle of 120° between the organic phase pipeline and the aqueous phase pipeline, an arrow-type mixing device with an angle of 240° between the organic phase pipeline and the aqueous phase pipeline, and a T-type mixing device with an angle of 180° between the organic phase pipeline and the aqueous phase pipeline. The total flow rate and the flow rate ratio of the organic phase to the aqueous phase in different mixing devices are shown in Table 6, and the performance of the obtained product is also shown in Table 6.

[0085] The results showed that in pH 4.0 citric acid-disodium hydrogen phosphate buffer, there were certain differences in the quality of nucleic acid nanolipid carriers prepared by different mixing equipment. Among them, the carriers prepared in the chip-type mixer had the best drug loading rate. When the N / P ratio was 3.44 / 1, the drug loading rate could still reach 96.89%. Compared with the traditional T-type mixing device, it has obvious advantages under low N / P ratio conditions.

[0086] Table 6:

[0087]

[0088] Example 7 Effect of using different buffer solutions in Y-type and T-type mixing devices on drug loading and recovery

[0089] The microfluidic multi-channel fluid mixing devices used in "3. Preparation of nucleic acid nanolipid carriers" in this embodiment are a Y-type mixing device and a T-type mixing device, respectively. The remaining parameters in "3. Preparation of nucleic acid nanolipid carriers" are the same as in Example 1, and "1. Preparation of organic phase" is the same as in Example 1. Except for the different types of buffer solutions and nucleic acid concentrations in "2. Preparation of aqueous phase", the remaining operations are the same as in Example 1.

[0090] The mixing device used in this embodiment includes: a Y-type mixing device with an angle of 120° between the organic phase pipeline and the aqueous phase pipeline and a T-type mixing device with an angle of 180° between the aqueous phase pipeline, the initial concentrations of siRNA are 120 μg / mL and 240 μg / mL, respectively, and the types of buffer solutions used in different mixing devices are shown in Tables 7-8, and the particle size, drug loading rate and recovery rate of the obtained nucleic acid nanolipid carrier are also shown in Tables 7-8. Among them, Table 7 is the parameters and effect data of the product prepared using the Y-type mixing device, and Table 8 is the parameters and effect data of the product prepared using the T-type mixing device.

[0091] The results showed that no matter which mixing device was used, the nucleic acid nanolipid carrier was optimal in pH 4.0 citric acid-disodium hydrogen phosphate buffer. In addition, in pH 4.0 citric acid-disodium hydrogen phosphate buffer, the drug loading rate and recovery rate of the nanolipid carrier samples prepared by the Y-type mixing device were higher than those of the traditional T-type mixing device, indicating that the mixing efficiency of the Y-type mixing device is higher than that of the T-type.

[0092] Table 7: Nanolipid carrier samples prepared using different buffer solutions in a Y-type mixing device

[0093]

[0094] Table 8: Nanolipid carrier samples prepared using different buffer solutions in a T-type mixing device

[0095]

[0096] Example 8 Effects of different angles on drug particle size and PDI in a Y-type mixing device

[0097] The operations of "1. preparing organic phase" and "2. preparing aqueous phase" in this embodiment are the same as those in Example 1, but the microfluidic multi-channel fluid mixing device and flow rate-related parameters used in "3. preparing nucleic acid nanolipid carrier" are different.

[0098] The mixing devices used in this embodiment include: a Y-type mixing device in which the angle between the organic phase pipeline and the aqueous phase pipeline is 120° and a Y-type mixing device in which the angle between the organic phase pipeline and the aqueous phase pipeline is 60°. The total flow rate and the flow rate ratio of the organic phase to the aqueous phase in different mixing devices, and the performance of the product are shown in Table 9.

[0099] Under the same total flow rate and flow rate ratio conditions, the 120° Y-type mixing device can obtain smaller particle size and PDI, which is more in line with the LNP medication requirements. At the same time, the particle size and PDI have a great influence on the stability of the LNP sample. The smaller the PDI, the better the LNP dispersion, and the less likely it is to flocculate and cause changes in properties.

[0100] In conclusion, under the same conditions, it is easier to obtain better LNP samples using a 120° Y-type mixing device, which improves the preparation efficiency of LNP.

[0101] Table 9:

[0102]

[0103] Example 9 Effects of different lipid types on drug loading and recovery

[0104] In this embodiment, in “1. Preparation of organic phase”, the ionizable phospholipids are replaced by DOTAP, ALC 0315 and SM102 respectively, and the rest of the operations in “1. Preparation of organic phase” and “2. Preparation of aqueous phase” are the same as those in Embodiment 3.

[0105] The microfluidic multi-channel fluid mixing devices used in "3. Preparation of nucleic acid nanolipid carriers" in this embodiment are a chip-type mixing device with an angle of 90° between the organic phase channel and the aqueous phase channel, an arrow-shaped mixing device with an angle of 240° between the organic phase channel and the aqueous phase channel, and a T-shaped mixing device with an angle of 180° between the organic phase channel and the aqueous phase channel. In addition, the total flow rate using the arrow-shaped mixing device and the T-shaped mixing device is 25 mL / min, and the remaining operations are the same as in Example 3. The specific parameters and effect data are shown in Tables 10-11.

[0106] The results showed that when LNP samples were prepared using different types of lipids, the nucleic acid nanolipid carriers prepared using arrow-shaped mixers and chip-shaped mixers had higher drug loading rates and recovery rates, and the drug loading rates and recovery rates of the nucleic acid nanolipid carriers prepared using T-shaped mixers were significantly improved.

[0107] Table 10:

[0108]

[0109] Table 11:

[0110]

[0111] From the above description, it can be seen that the above-mentioned embodiments of the present invention achieve the following technical effects: by controlling the structural parameters of the microfluidic device, such as the angle of the feed port, the operating conditions, and the type of buffer solution, the total flow rate and other parameters, it is possible to efficiently and stably generate nucleic acid nanolipid carriers with uniform particle size, significantly improving the encapsulation efficiency of the carrier and the stability of the nucleic acid.

[0112] In addition, this preparation method can also be mass-produced to meet the needs of clinical applications, providing strong technical support for the development of nucleic acid drugs. According to different nucleic acid types and treatment needs, the lipid composition and operating parameters can be flexibly adjusted to achieve personalized and customized nucleic acid drug preparation, which has broad application prospects and market potential.

[0113] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing a nucleic acid nanolipid carrier, characterized in that: The preparation method comprises: The total lipids are first mixed with an organic solvent to obtain an organic phase; performing a second mixing of the nucleic acid and the buffer solution to obtain an aqueous phase; Passing the organic phase and the aqueous phase into an organic phase pipeline and an aqueous phase pipeline respectively for third mixing to obtain the nucleic acid nanolipid carrier; Wherein, the organic phase pipeline and the aqueous phase pipeline are respectively the organic phase pipeline and the aqueous phase pipeline in a microfluidic multi-channel fluid mixing device, and the microfluidic multi-channel fluid mixing device includes: an arrow-shaped mixer, a chip-shaped mixer or a Y-shaped mixer; The chip-type mixer comprises two sample inlets and one sample outlet, the two sample inlets are arranged in parallel, the two sample inlets are connected to the same sample outlet through the organic phase pipeline and the aqueous phase pipeline respectively, the organic phase pipeline and the aqueous phase pipeline are continuously and cross-arranged, the organic phase pipeline and the aqueous phase pipeline are connected, and the sample inlet, the sample outlet, the organic phase pipeline and the aqueous phase pipeline are all arranged on a flat plate; The angle between the organic phase pipeline and the aqueous phase pipeline is θ, When the microfluidic multi-channel fluid mixing device is the arrow-shaped mixer, the angle θ is 200-300°, when the microfluidic multi-channel fluid mixing device is the chip-shaped mixer, the angle θ is 60-120°, and when the microfluidic multi-channel fluid mixing device is the Y-shaped mixer, the angle θ is 80-160°; The flow rate ratio of the organic phase to the aqueous phase is 1:1-9; When the microfluidic multi-channel fluid mixing device is the Y-shaped mixer or the arrow-shaped mixer, the total flow rate of the nucleic acid nanolipid carrier is 5-50 mL / min; When the microfluidic multi-channel fluid mixing device is the chip-type mixer, the total flow rate of the nucleic acid nanolipid carrier is 3-10 mL / min.

2. The preparation method according to claim 1, characterized in that: The total lipids include one or more of ionizable phospholipids, neutral phospholipids, cholesterol or pegylated phospholipids; The ionizable phospholipids include: [(4-hydroxybutyl) azadiyl] bis(hexane-6,1-diyl) bis(2-hexyldecanoate), 8-[(2-hydroxyethyl) (6-oxo-6-decyloxyhexyl) amino] octanoic acid (heptadecan-9-yl) ester, 4-(N,N-dimethylamino) butyric acid (dilinoleyl) methyl ester, N2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane or (2,3-dioleoyl-propyl)-trimethylammonium chloride or 1,1 '-[(2-{4-[2-({2-[bis(2-hydroxydodecyl)amino]ethyl}(2-hydroxydodecyl)amino)ethyl]piperazine-1-yl}ethyl) azadialkyl] bis(dodecane-2-ol) one or more; The neutral phospholipids include: one or more of 1,2-distearoyl-sn-glyceryl-3-phosphatidylcholine, 1,2-dioleoyl-sn-glyceryl-3-phosphatidylcholine, 1,2-dipalmitoyl-sn-glyceryl-3-phosphatidylcholine, 2-oleoyl-1-palmitoyl-sn-glyceryl-3-phosphatidylcholine, 1,2-dioleoyl-sn-glyceryl-3-phosphatidylethanolamine or 1,2-distearoyl-sn-glyceryl-3-phosphatidylethanolamine; The PEGylated phospholipids include one or more of 1,2-dimyristoyl-rac-glyceryl-3-methoxypolyethylene glycol, 1,2-distearoyl-rac-glyceryl-3-methoxypolyethylene glycol, 1,2-dipalmitoyl-rac-glyceryl-3-methoxypolyethylene glycol or 1,2-distearoyl-sn-glyceryl-3-phosphatidylethanolamine-methoxypolyethylene glycol; The organic solvent includes ethanol.

3. The preparation method according to claim 2, characterized in that: The total lipids include the ionizable phospholipids, the neutral phospholipids, the cholesterol and the PEGylated phospholipids, and in the organic phase, the concentration of the ionizable phospholipids is 1-10 mM; In the organic phase, the concentration of the neutral phospholipid is 0.5-5 mM; In the organic phase, the concentration of the PEGylated phospholipid is 0-2 mM; In the organic phase, the concentration of cholesterol is 2-5 mM.

4. The preparation method according to claim 3, characterized in that: In the total lipids, the molar fraction of the ionizable phospholipids is 30% to 60%, the molar fraction of the neutral phospholipids is 5% to 35%, the molar fraction of the cholesterol is 10% to 40%, and the molar fraction of the PEGylated phospholipids is 1% to 3%.

5. The preparation method according to claim 2, characterized in that: The buffer solution is selected from one or more of the following: citric acid buffer solution, phosphate buffer solution, amino acid buffer solution, Tris buffer solution or acetate buffer solution; The nucleic acid includes: siRNA, mRNA, circular RNA, microRNA, antisense nucleic acid or plasmid; The pH value of the buffer solution is 3 to 10; The concentration of the buffer solution is 5-500 mM.

6. The preparation method according to claim 2, wherein the molar ratio of the nucleic acid to the total lipids is 1:200-800.

7. The preparation method according to claim 1, characterized in that: The concentration of the nucleic acid is 0.01-500 μg / mL.

8. The preparation method according to claim 2, characterized in that: When the total lipids include the ionizable phospholipids, the molar ratio of the ionizable phospholipids to the N / P element in the nucleic acid is 2-20:

1.

9. The preparation method according to claim 1, characterized in that: The mixed product after the third mixing is subjected to ultrafiltration treatment, and an ultrafiltration tube is used for the ultrafiltration treatment, wherein the molecular weight cutoff of the ultrafiltration tube is 10-100KDa.

10. The nucleic acid nano-lipid carrier obtained by the preparation method of the nucleic acid nano-lipid carrier according to any one of claims 1 to 9 is characterized in that: The average particle size of the nucleic acid nanolipid carrier is 50-300nm. The nucleic acid nanolipid carrier according to claim 10 , wherein the distribution index (PDI) of the nucleic acid nanolipid carrier is 0.05-0.

4.

12. Use of the nucleic acid nano-lipid carrier obtained by the preparation method of the nucleic acid nano-lipid carrier according to any one of claims 1 to 9 in the preparation of nucleic acid drugs.

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