Gas-containing multivesicular lipid nanoparticles and their application and preparation method

Abstract

The present invention provides a gas-containing multivesicular lipid nanoparticle and its application and preparation method. The lipid nanoparticle is a multivesicular nanoparticle composed of lipid vesicles with water as the inner cavity and lipid vesicles with gas as the inner cavity. The lipid nanoparticle can be used as a delivery system for nucleic acids or small molecule chemical drugs, and the corresponding vaccine or pharmaceutical composition is prepared by rapidly mixing the raw materials under the conditions of ultrasound and surfactant. While delivering nucleic acids or small molecule chemical drugs, the lipid nanoparticle can also provide an oxygen-rich or hydrogen-rich environment with positive biological effects, thereby improving the delivery efficiency of nucleic acids or small molecule chemical drugs. In addition, applying ultrasound in vitro can achieve directional explosion of gas-containing vesicles, which is expected to further improve the bioavailability and targeting of nucleic acids or small molecule chemical drugs.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a gas-containing multivesicular lipid nanoparticle and a preparation method thereof, as well as use of the lipid nanoparticle as a nucleic acid or small molecule chemical drug delivery system. Background Art

[0002] With the rapid progress in the biomedical field, the proportion of biological preventive or therapeutic drugs in the entire pharmaceutical market continues to increase. Among them, the most representative biological drugs are nucleic acid vaccines / drugs. As a typical representative of nucleic acid vaccines / drugs, messenger RNA (mRNA) vaccines have shown great advantages over traditional vaccines in the fight against the new crown epidemic, leading the global biomedical research boom and having great application potential in the fields of tumor treatment, infectious disease prevention, protein replacement, and rare disease treatment.

[0003] The production process of mRNA vaccines / drugs mainly includes steps such as mRNA sequence design, synthesis, modification and delivery. Among them, efficient mRNA delivery technology is the most critical, which can enable structurally unstable and negatively charged mRNA to successfully pass through the equally negatively charged cell membrane into the cell. At present, the mainstream delivery system for mRNA is lipid nanoparticles, and both Pfizer / BioNTech and Morderna's mRNA COVID-19 vaccines are delivered using this system. Common lipid nanoparticles are nanoscale lipid vesicles formed by self-assembly of ionizable cationic lipids, neutral auxiliary phospholipids, cholesterol, and polyethylene glycol (PEG) phospholipids. Neutron scattering technology shows that the medium in this type of lipid vesicle is water (ACS Nano, 2023, 17, 979-990). However, although lipid nanoparticles have made significant progress as mRNA delivery systems in the fields of infectious disease prevention and tumor treatment and have been successfully applied in clinical practice, research results show that the proportion of mRNA successfully delivered to cells and expressed by lipid nanoparticles is less than 10%. Obviously, there is still huge room for optimization in the delivery efficiency of lipid nanoparticles, which is crucial to improving the effectiveness and safety of nucleic acid vaccines / drugs using lipid nanoparticles as delivery systems. Summary of the invention

[0004] In order to improve the delivery efficiency of lipid nanoparticles, the present invention provides a gas-containing multivesicular lipid nanoparticle and its application and preparation method.

[0005] First, the present invention provides a gas-containing multivesicular lipid nanoparticle, which is composed of ionizable cationic lipids and / or permanent cationic lipids, neutral auxiliary phospholipids, steroid compounds, polyethylene glycol (PEG) phospholipids, gas, and water; the lipid nanoparticle structure is a gas-containing multivesicular structure, which is composed of lipid vesicles with an inner cavity of water and lipid vesicles with an inner cavity of gas; wherein the lipid vesicles with an inner cavity of water are located at the center of the multivesicles, and the lipid vesicles with an inner cavity of gas are distributed at the edge of the lipid vesicles with an inner cavity of water.

[0006] In a preferred embodiment, the lipid nanoparticle is composed of a lipid vesicle with an inner cavity of water and one or more lipid vesicles with an inner cavity of gas distributed on the edge of the lipid vesicle.

[0007] In a preferred embodiment, the ionizable cationic lipid and / or permanent cationic lipid is one or more of 4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester (Dlin-MC3-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-1,3-dioxohexane (Dlin-KC2-DMA), 2,3-dioleoyloxypropyl-1-trimethylammonium bromide (DOTMA), (2,3-dioleoyl-propyl)-trimethylamine (DOTAP), heptadecan-9-yl-8-((2-hydroxyethyl)(6-oxo-6-((decyloxy)hexyl)amino)octanoate) (SM-102), and ((4-hydroxybutyl)azadiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315).

[0008] In a more preferred embodiment, the ionizable cationic lipid is one or more of Dlin-MC3-DMA, SM-102, and ALC-0315.

[0009] In a preferred embodiment, the neutral auxiliary phospholipid is one or more of 1,2-distearoyl-sn-glycerophosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), dipalmitoylphosphatidylcholine (DPPC), dimyristoylphosphatidylcholine (DMPC) or (2,3-dioleoyl-propyl)-trimethylamine sulfate (DOTAP).

[0010] In a more preferred embodiment, the neutral auxiliary phospholipid is one or both of DSPC and DOPE.

[0011] In a preferred embodiment, the steroidal compound is one or more of cholesterol, sitosterol, saposterol, campesterol, and stigmasterol.

[0012] In a more preferred embodiment, the steroid is preferably cholesterol.

[0013] In a preferred embodiment, the PEG lipid is one or more of distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000), 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000 (DMG-PEG2000), and methoxypolyethylene glycol ditetradecanoyl acetamide (ALC-0159).

[0014] In a more preferred embodiment, the PEG lipid is DMG-PEG2000.

[0015] In a preferred embodiment, the gas is one or more of oxygen, hydrogen and nitrogen.

[0016] In a preferred embodiment, excluding water and gas, the molar percentage of the ionizable cationic lipid and / or permanent cationic lipid is 40%-70%.

[0017] In a more preferred embodiment, excluding water and gas, the molar percentage of the ionizable cationic lipid and / or permanent cationic lipid is 45%-60%.

[0018] In a preferred embodiment, excluding water and gas, the molar percentage of the neutral auxiliary phospholipid is 2%-25%.

[0019] In a more preferred embodiment, excluding water and gas, the molar percentage of the neutral auxiliary phospholipid is 5%-20%.

[0020] In a preferred embodiment, the molar percentage of the steroidal compound is 20% to 60% when water and gas are excluded.

[0021] In a preferred embodiment, the molar percentage of the steroidal compound is preferably 30% to 50% when water and gas are excluded.

[0022] In a preferred embodiment, when water and gas are not taken into account, the molar percentage of the PEG phospholipid is 0.25%-5%.

[0023] In a more preferred embodiment, when water and gas are excluded, the molar percentage of the PEG phospholipid is preferably 0.8%-2%.

[0024] In a preferred embodiment, the percentage of water in the lipid nanoparticle volume is 20%-60%.

[0025] In a more preferred embodiment, the percentage of water in the volume of the lipid nanoparticles is 30%-55%.

[0026] In a preferred embodiment, the percentage of the gas in the volume of the lipid nanoparticles is 2%-20%.

[0027] In a more preferred embodiment, the percentage of the gas in the volume of the lipid nanoparticles is 5%-15%.

[0028] In a specific embodiment, excluding water and gas, the molar percentages of ionizable cationic lipids, neutral auxiliary phospholipids, steroids, and PEG phospholipids in the lipid nanoparticles are 50%, 10%, 38.5%, and 1.5%, respectively, and the percentages of water and gas in the volume of the lipid nanoparticles are 40% and 10%, respectively.

[0029] In a preferred embodiment, the average particle size of the lipid nanoparticles is 50-250 nm.

[0030] In a more preferred embodiment, the average particle size of the lipid nanoparticles is 60-150 nm.

[0031] Secondly, the present invention provides a vaccine or a pharmaceutical composition, which comprises the above-mentioned lipid nanoparticles and a pharmaceutically active component, wherein the lipid nanoparticles encapsulate the pharmaceutically active component.

[0032] In a preferred embodiment, the pharmaceutically active ingredient is a nucleic acid or a small molecule chemical drug.

[0033] In a preferred embodiment, the nucleic acid is a small interfering RNA (siRNA) or a messenger RNA (mRNA).

[0034] In a preferred embodiment, the mRNA is a linear non-self-replicating mRNA, a linear self-replicating mRNA or a circular mRNA.

[0035] In a preferred embodiment, the small molecule chemical drug includes one or more of doxorubicin, curcumin, paclitaxel, docetaxel, carotenoids, vitamin A, vitamin C, and vitamin D.

[0036] Finally, the present invention provides a process for preparing the vaccine or pharmaceutical composition, comprising the following steps:

[0037] S1. Solution configuration

[0038] (1) Dissolving ionizable cationic and / or permanent cationic lipids, neutral auxiliary phospholipids, steroids, PEG phospholipids, and surfactants in an organic solvent as an organic phase. The total concentration of lipids (including ionizable cationic and / or permanent cationic lipids, neutral auxiliary phospholipids, steroids, and PEG phospholipids) in the organic phase is 0.5-40 mg / mL, preferably 5-20 mg / mL; the surfactant is one or two of sodium dodecyl sulfate, Tween 20, Tween 60, and Tween 60, and the mass fraction of the surfactant in the organic phase is 0.01%-2%, preferably 0.1%-1%; the organic solvent is one or more of ethanol, acetonitrile, acetone, tetrahydrofuran, and N,N-dimethylformamide.

[0039] (2) When the active pharmaceutical ingredient is nucleic acid, the nucleic acid is dispersed in an acidic buffer solution with a pH of 3-6 as the aqueous phase. The acidic buffer solution includes one or both of an acetic acid-sodium acetate buffer solution and a citrate buffer solution; the concentration of the nucleic acid in the aqueous phase is 0.02-5 mg / mL, preferably 0.1-1 mg / mL.

[0040] (3) When the active component of the drug is a small molecule chemical drug, if the small molecule chemical drug is soluble in water, it is preferentially dissolved in water, and a buffer solution is used to control the pH value of the aqueous phase to 3-7 as the aqueous phase; if the small molecule chemical drug is insoluble in water, it is dissolved in an organic solvent together with an ionizable cationic lipid, a neutral auxiliary phospholipid, a steroid compound, and a PEG phospholipid as the organic phase, and pure water is used as the aqueous phase, and a buffer solution is used to control the pH value of the aqueous phase to 3-7. The buffer solution includes one or two of acetic acid-sodium acetate buffer solution, citrate buffer solution, and phosphate buffer solution; the concentration of the small molecule chemical drug in the aqueous phase or the organic phase is 0.02-300 mg / mL, preferably 0.1-180 mg / mL.

[0041] S2. Preparation of gas-rich solution

[0042] The water phase and the organic phase are purged with gas for 0.5-2 hours to obtain a gas-rich solution, namely a gas-rich water phase and a gas-rich organic phase, respectively.

[0043] S3, solution mixing

[0044] Under the action of ultrasound, the gas-rich aqueous phase and the gas-rich organic phase are quickly mixed in a predetermined volume ratio. In the presence of a surfactant, a large number of submicron cavitation bubbles escape and are encapsulated by lipid nanoparticles together with the active pharmaceutical ingredients to obtain a crude product; wherein the ultrasonic power is 1-1000W, preferably 5-200W; the ultrasonic frequency is 20KHz-1 MHz, preferably 20-100KHz; the ultrasonic treatment time is 0.01 seconds to 1 hour, preferably 0.2 seconds to 10 minutes; the volume ratio of the aqueous phase to the organic phase is 1:(0.2-50), preferably 1:(0.5-20).

[0045] S4. Product post-processing

[0046] First, a buffer solution is added to the crude product obtained in step S3 for dilution. Subsequently, the diluted crude product is subjected to solvent replacement and concentration using tangential flow technology. Finally, the concentrated product is subjected to aseptic treatment to obtain a final product. Wherein, the buffer solution is a phosphate buffer solution.

[0047] In a preferred experimental scheme, the preparation process of the nucleic acid vaccine or pharmaceutical composition of the present invention comprises the following steps:

[0048] S1. Solution configuration

[0049] (1) dissolving ionizable cations, neutral phospholipids, cholesterol, PEG phospholipids, and surfactants in an organic solvent as an organic phase;

[0050] (2) dispersing the nucleic acid into an acidic buffer solution with a pH of 3-6 as an aqueous phase; wherein the acidic buffer solution comprises one or both of an acetic acid-sodium acetate buffer solution and a citrate buffer solution;

[0051] S2. Preparation of gas-rich solution

[0052] The aqueous phase and the organic phase are purged with gas for 0.5-2 hours to obtain a gas-rich solution, namely a gas-rich aqueous phase solution and a gas-rich organic phase solution, respectively.

[0053] S3, solution mixing

[0054] Under the action of ultrasound, the gas-rich aqueous phase solution and the gas-rich organic phase solution are mixed at a volume ratio of 1:(0.2-50), preferably 1:(0.5-20). In the presence of a surfactant, a large number of submicron cavitation bubbles escape and are encapsulated by lipid nanoparticles together with nucleic acids to obtain a crude product;

[0055] S4. Product post-processing

[0056] First, a buffer solution is added to the crude product obtained in step S3 for dilution. Subsequently, the diluted crude product is subjected to solvent replacement and concentration using tangential flow technology. Finally, the concentrated product is subjected to aseptic treatment to obtain a final product.

[0057] In a preferred experimental scheme, the preparation process of the nucleic acid vaccine or drug combination of the present invention comprises the following steps:

[0058] S1. Solution configuration

[0059] (1) dissolving ionizable cations, neutral phospholipids, cholesterol, PEG phospholipids, and surfactants in an organic solvent as an organic phase;

[0060] (2) dispersing the nucleic acid into an acidic buffer solution with a pH of 3-6 as an aqueous phase; wherein the acidic buffer solution comprises one or both of an acetic acid-sodium acetate buffer solution and a citrate buffer solution;

[0061] S2. Preparation of gas-rich solution

[0062] The aqueous phase and the organic phase are purged with gas for 0.5-2 hours to obtain a gas-rich solution, namely a gas-rich aqueous phase solution and a gas-rich organic phase solution, respectively.

[0063] S3, solution mixing

[0064] Using ultrasound-assisted microfluidics technology, the gas-rich aqueous phase solution and the gas-rich organic phase solution are mixed at a volume ratio of 1:(0.2-50), preferably 1:(0.5-20). In the presence of a surfactant, a large number of submicron cavitation bubbles escape and are encapsulated by lipid nanoparticles together with nucleic acids to obtain a crude product;

[0065] S4. Product post-processing

[0066] First, a buffer solution is added to the crude product obtained in step S3 for dilution. Subsequently, the diluted crude product is subjected to solvent replacement and concentration using tangential flow technology. Finally, the concentrated product is subjected to aseptic treatment to obtain a final product.

[0067] In a preferred experimental scheme, the preparation process of the small molecule chemical drug combination of the present invention comprises the following steps:

[0068] S1. Solution configuration

[0069] (1) dissolving ionizable cationic and / or permanent cationic lipids, neutral phospholipids, cholesterol, PEG phospholipids, and surfactants in an organic solvent as an organic phase;

[0070] (2) dissolving doxorubicin in water and adjusting the pH to 7.4 with phosphate buffered saline as the aqueous phase;

[0071] S2. Preparation of gas-rich solution

[0072] The aqueous phase and the organic phase are purged with gas for 0.5-2 hours to obtain a gas-rich solution, namely a gas-rich aqueous phase solution and a gas-rich organic phase solution, respectively.

[0073] S3, solution mixing

[0074] Under the action of ultrasound, the gas-rich aqueous phase solution and the gas-rich organic phase solution are mixed at a volume ratio of 1:(0.2-50). In the presence of a surfactant, a large number of submicron cavitation bubbles escape and are encapsulated by lipid nanoparticles together with doxorubicin to obtain a crude product;

[0075] S4. Product post-processing

[0076] First, a buffer solution is added to the crude product obtained in step S3 for dilution. Subsequently, the diluted crude product is subjected to solvent replacement and concentration using tangential flow technology. Finally, the concentrated product is subjected to aseptic treatment to obtain a final product.

[0077] In a preferred experimental scheme, the preparation process of the small molecule chemical drug combination of the present invention comprises the following steps:

[0078] S1. Solution configuration

[0079] (1) dissolving ionizable cations, neutral phospholipids, cholesterol, PEG phospholipids, surfactants, and vitamin A in an organic solvent as an organic phase;

[0080] (2) using phosphate buffered saline (pH = 7.4) as the aqueous phase;

[0081] S2. Preparation of gas-rich solution

[0082] The aqueous phase and the organic phase are purged with gas for 0.5-2 hours to obtain a gas-rich solution, namely a gas-rich aqueous phase solution and a gas-rich organic phase solution, respectively.

[0083] S3, solution mixing

[0084] Under the action of ultrasound, the gas-rich aqueous phase solution and the gas-rich organic phase solution are mixed at a volume ratio of 1:(0.2-50). In the presence of a surfactant, a large number of submicron cavitation bubbles escape and are encapsulated by lipid nanoparticles together with vitamin A to obtain a crude product;

[0085] S4. Product post-processing

[0086] First, a buffer solution is added to the crude product obtained in step S3 for dilution. Subsequently, the diluted crude product is subjected to solvent replacement and concentration using tangential flow technology. Finally, the concentrated product is subjected to aseptic treatment to obtain a final product.

[0087] The present invention provides a novel lipid nanoparticle with a structure different from that of conventional lipid nanoparticles. The novel lipid nanoparticle is composed of ionizable cationic lipids and / or permanent cationic lipids, neutral auxiliary phospholipids, steroids, PEG phospholipids, gas, and water, and has a multivesicular structure, which is composed of lipid vesicles with water in the inner cavity and lipid vesicles with gas in the inner cavity. The lipid nanoparticle can be used as a delivery system for nucleic acids or small molecule chemical drugs. The corresponding vaccine or drug composition is prepared by rapidly mixing the raw materials under the conditions of ultrasound and the presence of a surfactant. The principle is as follows: a large number of submicron bubbles are generated under the conditions of ultrasound and the presence of a surfactant. While strengthening the self-loading process of ionizable cationic lipids, neutral auxiliary phospholipids, cholesterol, and PEG phospholipids, the submicron cavitation bubbles are wrapped by a phospholipid membrane together with water to form a multivesicular structure. In the process of cell uptake, the novel lipid nanoparticle can provide an oxygen-rich or hydrogen-rich environment with positive biological effects while delivering nucleic acids, thereby significantly improving the nucleic acid delivery efficiency. The novel lipid nanoparticle can also be used for the delivery of small molecule chemical drugs to improve the bioavailability of small molecule chemical drugs.

[0088] Compared with the prior art, the present invention has the following beneficial effects:

[0089] (1) The multivesicular lipid nanoparticles provided by the present invention, which are composed of lipid vesicles with water as the inner cavity and lipid vesicles with gas as the inner cavity, can provide an oxygen-rich or hydrogen-rich environment with positive biological effects while delivering nucleic acids or small molecule chemicals, thereby significantly improving the delivery efficiency of nucleic acids or small molecule chemicals.

[0090] (2) The lipid vesicles with gas in the inner cavity contained in the lipid nanoparticles provided by the present invention can be used as artificial cavitation cores to produce cavitation effect under ultrasonic radiation, causing expansion, compression or explosion, thereby enhancing the permeability of cell membranes and vascular systems. In addition, the jet generated by the cavitation effect releases energy in the cell, and the cell membrane will instantly open "sound holes", which are good channels for promoting the entry of active drug components into cells. Therefore, in the process of cell uptake of the lipid nanoparticles provided by the present invention, applying ultrasound in vitro can achieve directional explosion of gas-containing vesicles, which is expected to further improve the bioavailability and targeting of nucleic acids or small molecule chemical drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0091] Figure 1 , cryo-electron microscopy photograph of mRNA-lipid nanoparticles in Example 1;

[0092] Figure 2 , dynamic light scattering particle size distribution diagram of mRNA-lipid nanoparticles in Example 1;

[0093] Figure 3 , Statistical results of fluorescence microscopy observation of EYFP mRNA (intense yellow fluorescent protein mRNA) of lipid nanoparticles in Example 1;

[0094] Figure 4 , In vivo imaging observation of lipid nanoparticles delivering Luc-mRNA in vivo in Example 1.

[0095] Figure 5 , cryo-electron microscopy photograph of mRNA-lipid nanoparticles in Comparative Example 1;

[0096] Figure 6 , Dynamic light scattering particle size distribution diagram of mRNA-lipid nanoparticles in Comparative Example 1;

[0097] Figure 7 , In vivo imaging observation of Luc-mRNA delivery by lipid nanoparticles in Comparative Example 1. DETAILED DESCRIPTION

[0098] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme of the present invention will be further described in detail below in conjunction with the embodiments of the present invention and the accompanying drawings. Obviously, the described embodiments are only embodiments of a part of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without creative work should fall within the scope of protection of the present invention. It should be noted that the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict. The present invention will be described in detail below in conjunction with the embodiments.

[0099] Example 1

[0100] (1) Preparation of mRNA-lipid nanoparticles using ultrasound-assisted microfluidic technology

[0101] First, (2,3-dioleoyl-propyl)-trimethylamine (SM102) was selected as an ionizable cationic lipid, 1,2-distearoyl-sn-glycerophosphatidylcholine (DSPC) was selected as a neutral auxiliary phospholipid, cholesterol was selected as a steroid compound, and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000 (DMG-PEG2000) was selected as a PEG phospholipid. The molar percentages of SM102, DSPC, cholesterol, and DMG-PEG2000 were 50%, 10%, 38.5%, and 1.5%, respectively. The above lipid substances and Tween 80 were simultaneously dissolved in anhydrous ethanol as an organic phase solution, the total concentration of the lipid substances was 20 mg / mL, and the mass fraction of Tween 80 was 0.1%. For in vitro cell transfection experiments, the aqueous solution was prepared by dispersing EYFP mRNA (mRNA of strong yellow fluorescent protein) in citrate buffer (10 mM, pH = 4.0) to control the molar ratio of EYFP mRNA to lipids to be 1:15; for in vivo transfection experiments, the aqueous solution was prepared by dispersing Luc-mRNA (firefly luciferase mRNA) in citrate buffer (10 mM, pH = 4.0) to control the molar ratio of Luc-mRNA to lipids to be 1:15. Subsequently, the aqueous and organic phases were purged with sterile oxygen for 1 hour to obtain an oxygen-rich solution. The organic and aqueous phase solutions were rapidly mixed at room temperature using ultrasound-assisted microfluidics, with a reactor channel diameter of 1 mm, a volume flow ratio of the organic phase solution to the aqueous phase solution of 3:1, a total volume flow of 8 mL / min, an ultrasound power of 10 W, an ultrasound frequency of 20 KHz, and an ultrasound treatment time of 0.8 s. The obtained mRNA-lipid nanoparticle solution was quickly transferred to a 50mL ultrafiltration tube, and the obtained solution was diluted 10 times with 1×PBS10 buffer solution. Subsequently, centrifugation was performed at room temperature for 15 minutes, and the ultrafiltered mRNA-lipid nanoparticle solution was collected and fixed to a concentration of 160ug / mL with 1×PBS buffer solution. Finally, the sample was filtered with a 0.22μm polyethersulfone filter membrane and sterilized to obtain the final product. A series of labelings were performed on the Luc-mRNA-lipid nanoparticles: The morphology of the prepared mRNA-lipid nanoparticles was characterized by cryo-electron microscopy, and the results are shown in the figure. Figure 1 The cryo-electron microscopy images showed that the prepared lipid nanoparticles were multivesicular, consisting of lipid vesicles with water in the inner cavity and lipid vesicles with gas in the inner cavity. The average particle size and distribution of the prepared mRNA-lipid nanoparticles were characterized by dynamic light scattering. The results are as follows Figure 2 As shown, the prepared mRNA-lipid nanoparticles have a particle size of 98 nm and a polydispersity coefficient as low as 0.05. The mRNA encapsulation efficiency was measured using RiboGreen (Thermofisher), and the encapsulation efficiency was 95%.

[0102] (2) In vitro cell transfection

[0103] The EYFP-mRNA-lipid nanoparticles prepared in step (1) were tested for in vitro delivery efficiency on a cell model. A549 cells were used and 96-well plates were plated at a cell density of 30,000 cells / well. After 18 hours, 50 ng / mL of EYFP-mRNA-lipid nanoparticles were used for in vitro cell transfection experiments, with five replicate wells used in each group of experiments. After 24 hours, the transfection efficiency was detected using a flow cytometer and a fluorescence microscope (see Figure 3 The results of in vitro cell transfection experiments showed that the transfection rate of EYFP-mRNA reached 99.38% and the fluorescence intensity was greater than 10 6 It can be seen that the lipid nanoparticles provided by the present invention have a high delivery efficiency for EYFP-mRNA and are excellent mRNA delivery vectors.

[0104] (3) In vivo transfection experiment

[0105] The delivery efficiency of the Luc-mRNA-lipid nanoparticles prepared in step (1) was tested on a mouse animal model. The Luc-mRNA-lipid nanoparticles were injected into mice (BALB / c, 8 weeks old) by intravenous and intramuscular injection, respectively. The dosage of each mouse was 10 μg, and each group had three mice. Six hours later, the mice were injected with the luciferin substrate D-luciferin and imaged in vivo using an in vivo imaging device to measure the luminescence intensity of the mice and test the in vivo delivery efficiency of the nanoliposome particles (see Figure 4 ). From the in vivo delivery efficiency results, it can be seen that the nanoliposome particles provided by the present invention can well deliver Luc-mRNA, the delivery efficiency is very high, the mice survive well, and the safety is good.

[0106] Example 2

[0107] First, (2,3-dioleoyl-propyl)-trimethylamine (SM102) was selected as an ionizable cationic lipid, 1,2-distearoyl-sn-glycerophosphatidylcholine (DSPC) was selected as a neutral auxiliary phospholipid, cholesterol was selected as a steroid compound, and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000 (DMG-PEG2000) was selected as a PEG phospholipid. The molar percentages of SM102, DSPC, cholesterol, and DMG-PEG2000 were 40%, 20%, 38.5%, and 1.5%, respectively. The above lipid substances and Tween 80 were simultaneously dissolved in anhydrous ethanol as an organic phase solution, the total concentration of the lipid substances was 40 mg / mL, and the mass fraction of Tween 80 was 1%. The preparation process of the aqueous solution is to disperse EYFP mRNA (mRNA of strong yellow fluorescent protein) in citrate buffer (10mM, pH=4.0), and control the molar ratio of EYFP mRNA to lipid substances to be 1:15. Subsequently, the aqueous phase and the organic phase are purged with sterile hydrogen for 2 hours to obtain an oxygen-rich solution. At room temperature, the organic phase solution and the aqueous phase solution are quickly mixed using ultrasound-assisted microfluidics technology. The reactor channel diameter is 2 mm, the volume flow ratio of the organic phase solution to the aqueous phase solution is 3:1, the total volume flow rate is 16mL / min, the ultrasonic power is 20W, the ultrasonic frequency is 28KHz, and the ultrasonic treatment time is 1.2s. The obtained mRNA-lipid nanoparticle solution is quickly transferred to a 50mL ultrafiltration tube, and the obtained solution is diluted with 1×PBS10 buffer solution, and the dilution multiple is 10 times. Subsequently, at room temperature, centrifuge for 15min, collect the ultrafiltered mRNA-lipid nanoparticle solution, and dilute it to a concentration of 160ug / mL with 1×PBS buffer solution. Finally, the sample was filtered using a 0.22 μm polyethersulfone filter membrane and sterilized. Cryo-electron microscopy images showed that the prepared lipid nanoparticles were multivesicular, consisting of lipid vesicles with water in the inner cavity and lipid vesicles with gas in the inner cavity. The average particle size and distribution of the prepared mRNA-lipid nanoparticles were characterized by dynamic light scattering. The particle size of the prepared mRNA-lipid nanoparticles was 82 nm, and the polydispersity coefficient was as low as 0.06. The mRNA encapsulation efficiency was determined using RiboGreen (Thermofisher), and the encapsulation efficiency was 93%

[0108] Example 3

[0109] First, (2,3-dioleoyl-propyl)-trimethylamine (SM102) was selected as an ionizable cationic lipid, 1,2-distearoyl-sn-glycerophosphatidylcholine (DSPC) was selected as a neutral auxiliary phospholipid, cholesterol was selected as a steroid compound, and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000 (DMG-PEG2000) was selected as a PEG phospholipid. The molar percentages of SM102, DSPC, cholesterol, and DMG-PEG2000 were 40%, 10%, 49%, and 1%, respectively. The above lipid substances and Tween 80 were simultaneously dissolved in anhydrous ethanol as an organic phase solution, the total concentration of the lipid substances was 20 mg / mL, and the mass fraction of Tween 80 was 0.5%. The preparation process of the aqueous solution is to disperse EYFP mRNA (mRNA of strong yellow fluorescent protein) in citrate buffer (10mM, pH=4.0), and control the molar ratio of EYFP mRNA to lipid substances to be 1:12. Subsequently, the aqueous phase and organic phase are purged with sterile oxygen for 0.5 hours to obtain an oxygen-rich solution. At room temperature, the organic phase solution and the aqueous phase solution are quickly mixed using ultrasound-assisted microfluidics technology. The reactor channel diameter is 1 mm, the volume flow ratio of the organic phase solution to the aqueous phase solution is 3:1, the total volume flow rate is 16mL / min, the ultrasonic power is 30W, the ultrasonic frequency is 40KHz, and the ultrasonic treatment time is 0.5s. The obtained mRNA-lipid nanoparticle solution is quickly transferred to a 50mL ultrafiltration tube, and the obtained solution is diluted with 1×PBS10 buffer solution, and the dilution multiple is 10 times. Subsequently, at room temperature, centrifuge for 15min, collect the ultrafiltered mRNA-lipid nanoparticle solution, and dilute it to a concentration of 160ug / mL with 1×PBS buffer solution. Finally, the sample was filtered using a 0.22 μm polyethersulfone filter membrane and sterilized. Cryo-electron microscopy images showed that the prepared lipid nanoparticles were multivesicular, consisting of lipid vesicles with water in the inner cavity and lipid vesicles with gas in the inner cavity. The average particle size and distribution of the prepared mRNA-lipid nanoparticles were characterized by dynamic light scattering. The particle size of the prepared mRNA-lipid nanoparticles was 75 nm, and the polydispersity coefficient was as low as 0.05. The mRNA encapsulation efficiency was determined using RiboGreen (Thermofisher), and the encapsulation efficiency was 94%

[0110] Example 4

[0111] First, 4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester (Dlin-MC3-DMA) was selected as an ionizable cationic lipid, 1,2-distearoyl-sn-glycerophosphatidylcholine (DSPC) was selected as a neutral auxiliary phospholipid, cholesterol was selected as a steroid compound, and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000 (DMG-PEG2000) was selected as a PEG phospholipid. The molar percentages of SM102, DSPC, cholesterol, and DMG-PEG2000 were 60%, 20%, 29%, and 1%, respectively. The above lipid substances and Tween 80 were dissolved in anhydrous ethanol at the same time as an organic phase solution, the total concentration of the lipid substances was 20 mg / mL, and the mass fraction of Tween 80 was 0.1%. The preparation process of the aqueous solution is to disperse EYFP mRNA (mRNA of strong yellow fluorescent protein) in citrate buffer (10mM, pH=4.0), and control the molar ratio of EYFP mRNA to lipid substances to be 1:15. Subsequently, the aqueous phase and the organic phase are purged with sterile oxygen for 1 hour to obtain an oxygen-rich solution. At room temperature, the organic phase solution and the aqueous phase solution are quickly mixed using ultrasound-assisted microfluidics technology. The volume flow ratio of the organic phase solution to the aqueous phase solution is 3:1, the total volume flow rate is 16mL / min, the ultrasonic power is 10W, the ultrasonic frequency is 20KHz, and the ultrasonic treatment time is 1.2s. The obtained mRNA-lipid nanoparticle solution is quickly transferred to a 50mL ultrafiltration tube, and the obtained solution is diluted with 1×PBS10 buffer solution, and the dilution multiple is 10 times. Subsequently, at room temperature, centrifuge for 15min, collect the ultrafiltered mRNA-lipid nanoparticle solution, and dilute it to a concentration of 160ug / mL with 1×PBS buffer solution. Finally, the sample was filtered using a 0.22 μm polyethersulfone filter membrane and sterilized. Cryo-electron microscopy images showed that the prepared lipid nanoparticles were multivesicular, consisting of lipid vesicles with water in the inner cavity and lipid vesicles with gas in the inner cavity. The average particle size and distribution of the prepared mRNA-lipid nanoparticles were characterized by dynamic light scattering. The particle size of the prepared mRNA-lipid nanoparticles was 90 nm, and the polydispersity coefficient was as low as 0.05. The mRNA encapsulation efficiency was determined using RiboGreen (Thermofisher), and the encapsulation efficiency was 96%

[0112] Example 5

[0113] First, 4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester (Dlin-MC3-DMA) was selected as an ionizable cationic lipid, 1,2-distearoyl-sn-glycerophosphatidylcholine (DSPC) was selected as a neutral auxiliary phospholipid, cholesterol was selected as a steroid compound, and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000 (DMG-PEG2000) was selected as a PEG phospholipid. The molar percentages of SM102, DSPC, cholesterol, and DMG-PEG2000 were 60%, 20%, 29%, and 1%, respectively. The above lipid substances, Tween 80, and vitamin D were dissolved in anhydrous ethanol at the same time as the organic phase solution. The total concentration of lipid substances was 20 mg / mL, the mass fraction of Tween was 0.1%, and the concentration of vitamin D was 100 mg / mL. The aqueous phase was a citrate buffer (10 mM, pH = 4.0). Subsequently, the aqueous phase and the organic phase were purged with sterile oxygen for 0.5 hours to obtain an oxygen-rich solution. At room temperature, the organic phase solution and the aqueous phase solution were quickly mixed using ultrasound-assisted microfluidics technology. The volume flow ratio of the organic phase solution to the aqueous phase solution was 3:1, the total volume flow rate was 20 mL / min, the ultrasonic power was 10 W, the ultrasonic frequency was 20 KHz, and the ultrasonic treatment time was 0.3 s. The obtained vitamin D-lipid nanoparticle solution was quickly transferred to a 50 mL ultrafiltration tube, and the obtained solution was diluted with 1×PBS10 buffer solution by 10 times. Subsequently, at room temperature, centrifuged for 15 minutes, the ultrafiltered vitamin D-lipid nanoparticle solution was collected, and the volume was fixed to a concentration of 160 ug / mL with 1×PBS buffer solution. Finally, the sample was filtered with a 0.22 μm polyethersulfone filter membrane for sterilization. Cryo-electron microscopy photos showed that the prepared lipid nanoparticles were multivesicular, consisting of lipid vesicles with water in the inner cavity and lipid vesicles with gas in the inner cavity. The average particle size and distribution of the prepared vitamin D-lipid nanoparticles were characterized by dynamic light scattering. The particle size of the prepared vitamin D-lipid nanoparticles was 75 nm, and the polydispersity coefficient was as low as 0.05.

[0114] Comparative Example 1

[0115] (1) Preparation of mRNA-lipid nanoparticles using microfluidic technology

[0116] First, (2,3-dioleoyl-propyl)-trimethylamine (SM102) was selected as an ionizable cationic lipid, 1,2-distearoyl-sn-glycerophosphatidylcholine (DSPC) was selected as a neutral auxiliary phospholipid, cholesterol was selected as a steroid compound, and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000 (DMG-PEG2000) was selected as a PEG phospholipid. The molar percentages of SM102, DSPC, cholesterol, and DMG-PEG2000 were 50%, 10%, 38.5%, and 1.5%, respectively. The above lipid substances and Tween 80 were simultaneously dissolved in anhydrous ethanol as an organic phase solution, the total concentration of the lipid substances was 20 mg / mL, and the mass fraction of Tween 80 was 0.1%. The configuration process of the aqueous solution is as follows: Luc-mRNA (firefly luciferase mRNA) is dispersed in a citrate buffer (10mM, pH=4.0), and the molar ratio of Luc-mRNA to lipid substances is controlled to be 1:15. Subsequently, the aqueous phase and the organic phase are purged with sterile oxygen for 1 hour to obtain an oxygen-rich solution. The organic phase solution and the aqueous phase solution are quickly mixed using microfluidics at room temperature. The geometric dimensions and configuration of the reactor channel are the same as those in Example 1. The volume flow ratio of the organic phase solution to the aqueous phase solution is 3:1, and the total volume flow rate is 8mL / min. The obtained mRNA-lipid nanoparticle solution is quickly transferred to a 50mL ultrafiltration tube, and the obtained solution is diluted with a 1×PBS10 buffer solution, and the dilution multiple is 10 times. Subsequently, at room temperature, centrifuge for 15min, collect the ultrafiltered mRNA-lipid nanoparticle solution, and dilute to a concentration of 160ug / mL with a 1×PBS buffer solution. Finally, the sample was filtered through a 0.22 μm polyethersulfone filter membrane and sterilized to obtain the final product. The morphology of the prepared mRNA-lipid nanoparticles was characterized by cryo-electron microscopy. Figure 5 The cryo-electron microscopy images showed that the prepared lipid nanoparticles were in the form of single vesicles with water inside. The average particle size and distribution of the prepared mRNA-lipid nanoparticles were characterized by dynamic light scattering. The results were as follows Figure 6 As shown, the prepared mRNA-lipid nanoparticles have a particle size of 115 nm and a polydispersity coefficient of 0.12. The mRNA encapsulation efficiency was measured using RiboGreen (Thermofisher), and the encapsulation efficiency was 85%.

[0117] (2) In vivo transfection experiment

[0118] The delivery efficiency of the Luc-mRNA-lipid nanoparticles prepared in step (1) was tested on a mouse animal model. The Luc-mRNA-lipid nanoparticles were injected into mice (BALB / c, 8 weeks old) by intravenous and intramuscular injection, respectively. The dosage of each mouse was 10 μg, and each group had three mice. Six hours later, the mice were injected with the luciferin substrate D-luciferin and imaged in vivo using an in vivo imaging device to measure the luminescence intensity of the mice and test the in vivo delivery efficiency of the nanoliposome particles (see Figure 7 ). As can be seen from the figure, the in vivo delivery efficiency of the single vesicular lipid nanoparticles without gas is significantly lower than that of the gas-containing multivesicular lipid nanoparticles prepared in Example 1.

Claims

1. A vaccine or pharmaceutical composition, characterized in that comprising lipid nanoparticles and a pharmaceutically active component; The lipid nanoparticles are gas-containing multivesicular lipid nanoparticles, and have a multivesicular structure. The multivesicles are composed of lipid vesicles with water as the inner cavity and lipid vesicles with gas as the inner cavity. The lipid vesicles with water as the inner cavity are located at the center of the multivesicles, and the lipid vesicles with gas as the inner cavity are distributed at the edge of the lipid vesicles with water as the inner cavity. The gas is oxygen. The active component of the drug is a nucleic acid or a small molecule chemical drug; The vaccine or pharmaceutical composition, the preparation process comprises the following steps: S1. Solution configuration (1) dissolving ionizable cationic and / or permanent cationic lipids, neutral auxiliary phospholipids, steroids, polyethylene glycol phospholipids, and surfactants in an organic solvent as an organic phase; (2) When the active ingredient of the drug is nucleic acid, the nucleic acid is dispersed in an acidic buffer solution with a pH of 3-6 as the aqueous phase; (3) When the active ingredient of the drug is a small molecule chemical drug, if the small molecule chemical drug is soluble in water, it is preferentially dissolved in water, and a buffer solution is used to control the pH value of the aqueous phase to 3-7 as the aqueous phase; if the small molecule chemical drug is insoluble in water, it is dissolved in an organic solvent simultaneously with ionizable cationic and / or permanent cationic lipids, neutral auxiliary phospholipids, steroidal compounds, and polyethylene glycol phospholipids as the organic phase, and pure water is used as the aqueous phase, and a buffer solution is used to control the pH value of the aqueous phase to 3-7; S2. Preparation of gas-rich aqueous phase and organic phase Purging the aqueous phase and the organic phase with gas for 0.5-2 hours to obtain a gas-rich aqueous phase and an organic phase, respectively; S3, solution mixing Under the action of ultrasound, the gas-rich organic phase and the gas-rich aqueous phase are quickly mixed at a predetermined volume ratio, and in the presence of a surfactant, a large number of submicron cavitation bubbles are formed, which are encapsulated by lipid nanoparticles together with the active pharmaceutical ingredients to obtain a crude product; S4. Product post-processing First, a buffer solution is added to the crude product obtained in step S3 for dilution; then, the diluted crude product is subjected to solvent replacement and concentration using tangential flow technology; finally, the concentrated product is subjected to aseptic treatment to obtain a final product.

2. The vaccine or pharmaceutical composition according to claim 1, characterized in that: The ionizable cationic lipid and / or permanent cationic lipid is one or more of 4-(N, N-dimethylamino)butyric acid (dilinoleyl) methyl ester, 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-1,3-dioxohexane, 2,3-dioleoyloxypropyl-1-trimethylamine bromide, and (2,3-dioleoyl-propyl)-trimethylamine; The neutral auxiliary phospholipid is one or more of 1,2-distearoyl-sn-glycerophosphatidylcholine, dioleoylphosphatidylcholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, dipalmitoylphosphatidylcholine, and dimyristoylphosphatidylcholine; The steroidal compound is one or more of cholesterol, sitosterol, saposterol, campesterol, and stigmasterol; The polyethylene glycol lipid is one or more of distearoyl phosphatidylethanolamine-polyethylene glycol 2000, 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol 2000, and methoxy polyethylene glycol ditetradecyl acetamide.

3. The vaccine or pharmaceutical composition according to claim 1, characterized in that: Lipid nanoparticles, excluding water and gas, wherein the molar percentage of the ionizable cationic lipid and / or permanent cationic lipid is 40%-70%; When water and gas are not counted, the molar percentage of the neutral auxiliary phospholipid is 2%-25%; When water and gas are not counted, the molar percentage of the steroidal compound is 20%-49%; When water and gas are not taken into account, the molar percentage of the polyethylene glycol phospholipid is 0.25%-5%.

4. The vaccine or pharmaceutical composition according to claim 3, characterized in that: Lipid nanoparticles, excluding water and gas, wherein the molar percentage of the ionizable cationic lipid and / or permanent cationic lipid is 45%-60%; When water and gas are not counted, the molar percentage of the neutral auxiliary phospholipid is 5%-20%; When water and gas are not counted, the molar percentage of the steroidal compound is 30%-49%; When water and gas are not taken into account, the molar percentage of the polyethylene glycol phospholipid is 0.8%-2%.

5. The vaccine or pharmaceutical composition according to claim 1, characterized in that: The water accounts for 20%-60% of the volume percentage of the lipid nanoparticles; The gas accounts for 2%-20% of the volume fraction of the lipid nanoparticles.

6. The vaccine or pharmaceutical composition according to claim 5, characterized in that: The water accounts for 30%-55% of the volume percentage of the lipid nanoparticles; The gas accounts for 5%-15% of the volume fraction of the lipid nanoparticles.

7. The vaccine or pharmaceutical composition according to claim 1, characterized in that: The average particle size of lipid nanoparticles is 50-250 nm.

8. The vaccine or pharmaceutical composition according to claim 7, characterized in that: The average particle size of lipid nanoparticles is 60-150 nm.

9. The vaccine or pharmaceutical composition according to claim 1, characterized in that: The nucleic acid is small interfering RNA or messenger RNA; the small molecule chemical drug includes one or more of doxorubicin, curcumin, paclitaxel, docetaxel, carotenoids, vitamin A, vitamin C, and vitamin D.

10. The vaccine or pharmaceutical composition according to claim 9, characterized in that: The messenger RNA is a linear non-self-replicating messenger RNA, a linear self-replicating messenger RNA or a circular messenger RNA.

11. The vaccine or pharmaceutical composition according to claim 1, characterized in that In step S1, the organic solvent is one or more of ethanol, acetonitrile, acetone, tetrahydrofuran, and N,N-dimethylformamide; the total concentration of ionizable cationic and / or permanent cationic lipids, neutral auxiliary phospholipids, steroids, and PEG phospholipids in the organic phase is 0.5-40 mg / mL; the surfactant is one or two of sodium dodecyl sulfate, Tween 20, Tween 60, and Tween 60, and the mass fraction of the surfactant in the organic phase is 0.01%-2%; In step S2, when the active pharmaceutical ingredient is a nucleic acid, the acidic buffer solution includes one or two of an acetic acid-sodium acetate buffer solution and a citrate buffer solution; the concentration of the nucleic acid in the aqueous phase is 0.02-5 mg / mL; when the active pharmaceutical ingredient is a small molecule chemical drug, the buffer solution includes one or two of an acetic acid-sodium acetate buffer solution, a citrate buffer solution, and a phosphate buffer solution; the concentration of the small molecule chemical drug in the aqueous phase or the organic phase is 0.02-300 mg / mL; In step S3, the ultrasonic power is 1-1000 W; the ultrasonic frequency is 20 KHz-1 MHz; the ultrasonic treatment time is 0.01 second to 1 hour; the volume ratio of the aqueous phase to the organic phase is 1:(0.2-50); In step S4, the buffer solution is a phosphate buffer solution.

12. The vaccine or pharmaceutical composition according to claim 11, characterized in that In step S1, the total concentration of ionizable cationic and / or permanent cationic lipids, neutral auxiliary phospholipids, steroids, and PEG phospholipids in the organic phase is 5-20 mg / mL; the mass fraction of the surfactant in the organic phase is 0.1%-1%; In step S2, when the active pharmaceutical ingredient is nucleic acid, the concentration of the nucleic acid in the aqueous phase is 0.1-1 mg / mL; when the active pharmaceutical ingredient is a small molecule chemical drug, the concentration of the small molecule chemical drug in the aqueous phase or the organic phase is 0.1-180 mg / mL; In step S3, the ultrasonic power is 5-200 W; the ultrasonic frequency is 20-100 KHz; the ultrasonic treatment time is 0.2 seconds to 10 minutes; and the volume ratio of the aqueous phase to the organic phase is 1:(0.5-20).

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