CN116807997A - Gas-containing multivesicular lipid nanoparticle, application and preparation method thereof - Google Patents

Gas-containing multivesicular lipid nanoparticle, application and preparation method thereof Download PDF

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CN116807997A
CN116807997A CN202310853746.6A CN202310853746A CN116807997A CN 116807997 A CN116807997 A CN 116807997A CN 202310853746 A CN202310853746 A CN 202310853746A CN 116807997 A CN116807997 A CN 116807997A
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lipid
gas
water
phospholipid
buffer solution
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陈光文
刘志凯
杨梅
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Dalian Institute of Chemical Physics of CAS
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    • A61K9/0002—Galenical forms characterised by the drug release technique; Application systems commanded by energy
    • A61K9/0009—Galenical forms characterised by the drug release technique; Application systems commanded by energy involving or responsive to electricity, magnetism or acoustic waves; Galenical aspects of sonophoresis, iontophoresis, electroporation or electroosmosis

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Abstract

The invention provides a gas-containing multivesicular lipid nanoparticle, and an application and a preparation method thereof. The lipid nanoparticle is a multi-vesicle nanoparticle composed of a lipid vesicle with a water inner cavity and a lipid vesicle with a gas inner cavity. The lipid nanoparticle can be used as a delivery system for nucleic acid or small molecule chemical drugs, and the corresponding vaccine or pharmaceutical composition is prepared by rapidly mixing raw materials in the presence of ultrasound and a surfactant. The lipid nanoparticle can provide an environment with forward biological effects such as oxygen enrichment or hydrogen enrichment while delivering nucleic acid or small molecular chemical drugs, and improves the delivery efficiency of the nucleic acid or the small molecular chemical drugs. In addition, the directional blasting of the air sac-containing bubbles can be realized by applying ultrasound in vitro, and the bioavailability and targeting of nucleic acid or small molecule chemical drugs are expected to be further improved.

Description

Gas-containing multivesicular lipid nanoparticle, application and preparation method thereof
Technical Field
The invention relates to the technical field of biological medicine, in particular to a gas-containing multivesicular lipid nanoparticle, a preparation method and application of the lipid nanoparticle as a nucleic acid or small molecule chemical drug delivery system.
Background
With the rapid progress in the biomedical field, the proportion of bio-preventive or therapeutic drugs in the whole pharmaceutical market is increasing. Among these, the most representative biopharmaceuticals are nucleic acid vaccines/drugs. As a typical representation of nucleic acid vaccines/drugs, messenger ribonucleic acid (mRNA) vaccines have great potential for application in the fields of tumor treatment, infectious disease prevention, protein substitution, rare disease treatment and the like, because they exhibit great advantages over traditional vaccines in combating new coronary epidemic situations, leading research hotness in the field of biological medicine worldwide.
The production process of mRNA vaccine/medicine mainly includes the steps of mRNA sequence design, synthesis, modification and delivery. Among them, the most critical is the efficient mRNA delivery technique, which allows structurally unstable and negatively charged mRNA to successfully cross the same negatively charged cell membrane into the cell. Currently, the predominant delivery system for mRNA is lipid nanoparticle, and both the new corona mRNA vaccines of pyroxene/BioNTech and Morderna are delivered using this system. Common lipid nanoparticles are nanoscale lipid vesicles formed by self-assembly from ionizable cationic lipids, neutral helper phospholipids, cholesterol, polyethylene glycol (PEG) phospholipids. Neutron scattering techniques showed that the lipid vesicle internal medium was water (ACS Nano,2023,17,979-990). However, although lipid nanoparticles have made remarkable progress as mRNA delivery systems in the fields of infectious disease prevention, tumor treatment, etc., and have been successfully applied to clinic, the results of studies indicate that the proportion of mRNA successfully delivered to cells and expressed by lipid nanoparticles is less than 10%. Clearly, there is still a great room for optimization of the delivery efficiency of lipid nanoparticles, which is crucial to improve the effectiveness and safety of nucleic acid vaccines/drugs with lipid nanoparticles as delivery system.
Disclosure of Invention
In order to improve the delivery efficiency of the lipid nanoparticle, the invention provides an air-containing multivesicular lipid nanoparticle, and an application and a preparation method thereof.
Firstly, the invention provides a gas-containing multivesicular lipid nanoparticle which consists of ionizable cationic lipid and/or permanent cationic lipid, neutral auxiliary phospholipid, steroid compound, polyethylene glycol (PEG) phospholipid, gas and water; the lipid nanoparticle structure is in the shape of a gas-containing multivesicular, and consists of a lipid vesicle with a water inner cavity and a lipid vesicle with a gas inner cavity; wherein, the lipid vesicle with the inner cavity of water is positioned in the center of the multi-vesicle, and the lipid vesicle with the inner cavity of gas is distributed at the edge of the lipid vesicle with the inner cavity of water.
In a preferred embodiment, the lipid nanoparticle is composed of one lipid vesicle with water in its lumen and one or more lipid vesicles with gas in its lumen distributed at its edge.
In a preferred embodiment, the ionizable and/or permanent cationic lipid is one or more of 4- (N, N-dimethylamino) butanoic acid (diiodo) methyl ester (Dlin-MC 3-DMA), 2-diiodo-4- (2-dimethylaminoethyl) -1, 3-dioxane (Dlin-KC 2-DMA), 2, 3-dioleoyloxypropyl-1-bromotrimethylamine (DOTMA), (2, 3-dioleoyl-propyl) -trimethylamine (DOTAP), heptadecane-9-yl-8- ((2-hydroxyethyl) (6-oxo-6- ((decyloxy) hexyl) amino) octanoate) (SM-102), ((4-hydroxybutyl) azadiyl) bis (hexane-6, 1-diyl) bis (2-hexyldecanoate) (ALC-0315).
In a more preferred embodiment, the ionizable cationic lipid is one or more of Dlin-MC3-DMA, SM-102, ALC-0315.
In a preferred embodiment, the neutral helper phospholipid is one or more of 1, 2-distearoyl-sn-glycerophosphorylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), dipalmitoyl phosphatidylcholine (DPPC), dimyristoyl phosphatidylcholine (DMPC), or (2, 3-dioleoyl-propyl) -trimethylamine sulfate (DOTAP).
In a more preferred embodiment, the neutral helper phospholipid is one or both of DSPC or DOPE.
In a preferred embodiment, the steroid is one or more of cholesterol, sitosterol, yasterol, campesterol, stigmasterol.
In a more preferred embodiment, the steroid is preferably cholesterol.
In a preferred embodiment, the PEG lipid is one or more of distearoyl phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG 2000), 1, 2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000 (DMG-PEG 2000), methoxypolyethylene glycol ditetradecylacetamide (ALC-0159).
In a more preferred embodiment, the PEG lipid is DMG-PEG2000.
In a preferred embodiment, the gas is one or more of oxygen, hydrogen, nitrogen.
In a preferred embodiment, the mole percent of the ionizable cationic lipid and/or permanent cationic lipid is 40% -70% when water and gas are not counted.
In a more preferred embodiment, the mole percent of the ionizable cationic lipid and/or permanent cationic lipid is 45% -60% when water and gas are not counted.
In a preferred embodiment, the mole percent of neutral helper phospholipid is 2% -25% when water and gas are not counted.
In a more preferred embodiment, the mole percent of neutral helper phospholipid is 5% to 20% when water and gas are not counted.
In a preferred embodiment, the molar percentage of the steroid is 20% -60% without water and gas.
In a preferred embodiment, the molar percentage of the steroid is preferably 30% -50% when water and gas are not counted.
In a preferred embodiment, the PEG phospholipid is present in a mole percentage of 0.25% -5% when water and gas are not counted,
in a more preferred embodiment, the PEG phospholipid is preferably present in a mole percentage of 0.8% -2% when water and gas are not counted.
In a preferred embodiment, the percentage of water by volume of the lipid nanoparticle is 20% -60%.
In a more preferred embodiment, the percentage of water is 30% -55% by volume of the lipid nanoparticle.
In a preferred embodiment, the gas comprises 2% -20% by volume of the lipid nanoparticle.
In a more preferred embodiment, the gas comprises from 5% to 15% by volume of the lipid nanoparticle.
In a specific embodiment, the mole percentages of the ionizable cationic lipid, neutral helper phospholipid, steroid, PEG phospholipid in the lipid nanoparticle are 50%, 10%, 38.5%, 1.5%, respectively, when water and gas are not counted, and the percentages of water and gas are 40% and 10%, respectively, by volume of the lipid nanoparticle.
In a preferred embodiment, the lipid nanoparticle has an average particle size of 50-250nm.
In a more preferred embodiment, the lipid nanoparticle has an average particle size of 60-150nm.
Next, the present invention provides a vaccine or pharmaceutical composition comprising the above lipid nanoparticle and a pharmaceutically active component, the lipid nanoparticle encapsulating the pharmaceutically active component.
In a preferred embodiment, the pharmaceutically active component is a nucleic acid or a small molecule chemical.
In a preferred embodiment, the nucleic acid is a small interfering RNA (siRNA) or a messenger RNA (mRNA).
In a preferred embodiment, the mRNA is a linear non-self-replicating mRNA, a linear self-replicating mRNA, or a circular mRNA.
In a preferred embodiment, the small molecule chemical comprises one or more of doxorubicin, curcumin, paclitaxel, docetaxel, carotenoids, vitamin a, vitamin C, vitamin D.
Finally, the invention provides a preparation process of the vaccine or the pharmaceutical composition, which comprises the following steps:
s1, solution configuration
(1) The ionizable cation and/or permanent cation lipid, neutral auxiliary phospholipid, steroid, PEG phospholipid, and surfactant are dissolved in organic solvent to obtain organic phase. Wherein the total concentration of lipids (including ionizable and/or permanent cationic lipids, neutral helper phospholipids, steroids, PEG phospholipids) in the organic phase is 0.5-40mg/mL, preferably 5-20mg/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.
(2) When the pharmaceutically active component is a nucleic acid, the nucleic acid is dispersed into an acidic buffer solution having ph=3-6 as an aqueous phase. Wherein the acidic buffer solution comprises one or two of acetic acid-sodium acetate buffer solution and citrate buffer solution; the concentration of the nucleic acid in the aqueous phase is 0.02-5mg/mL, preferably 0.1-1mg/mL.
(3) When the active component of the medicine is a small molecular chemical medicine, if the small molecular chemical medicine is soluble in water, the small molecular chemical medicine is preferentially dissolved in the water, and the pH value of the water phase is controlled to be 3-7 by using a buffer solution to serve as the water phase; if the small molecular chemical is insoluble in water, the small molecular chemical is dissolved in an organic solvent together with ionizable cationic lipid, neutral auxiliary phospholipid, steroid and PEG phospholipid, and the organic phase is pure water, and the pH value of the water phase is controlled to be 3-7 by using a buffer solution. Wherein the buffer solution comprises one or two of acetic acid-sodium acetate buffer solution, citrate buffer solution and phosphate buffer solution; the concentration of the small molecule chemical in the aqueous phase or the organic phase is 0.02-300mg/mL, preferably 0.1-180mg/mL.
S2, preparation of gas-rich solution
And (3) purging the water phase and the organic phase for 0.5-2 hours by using gas to obtain a gas-rich solution, namely a gas-rich water phase and a gas-rich organic phase respectively.
S3, mixing the solutions
Under the action of ultrasound, rapidly mixing the gas-rich water phase and the gas-rich organic phase in a preset volume ratio, and encapsulating a large amount of submicron cavitation bubbles, together with the active pharmaceutical ingredients, by lipid nanoparticles in the presence of a surfactant to obtain a crude product; wherein the ultrasonic power is 1-1000W, preferably 5-200W; the ultrasonic frequency is 20KHz-1MHz, preferably 20-100KHz; the sonication time is from 0.01 seconds to 1 hour, preferably from 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).
S4, post-treatment of products
First, a buffer solution is added to the crude product obtained in step S3 for dilution. The diluted crude product is then subjected to solvent displacement and concentration using tangential flow techniques. And finally, carrying out aseptic treatment on the concentrated product to obtain a final product. Wherein the buffer solution is phosphate buffer solution.
In a preferred embodiment, the process for preparing the nucleic acid vaccine or pharmaceutical composition of the present invention comprises the steps of:
s1, solution configuration
(1) Dissolving ionizable cations, neutral phospholipids, cholesterol, PEG phospholipids and surfactant in an organic solvent to obtain an organic phase;
(2) Dispersing nucleic acid into an acidic buffer solution with ph=3-6 as an aqueous phase; wherein the acidic buffer solution comprises one or two of acetic acid-sodium acetate buffer solution and citrate buffer solution;
s2, preparation of gas-rich solution
And (3) purging the water phase and the organic phase for 0.5-2 hours by using gas to obtain gas-rich solution, namely gas-rich water phase solution and gas-rich organic phase solution respectively.
S3, mixing the solutions
Under the action of ultrasound, mixing the gas-rich aqueous phase solution and the gas-rich organic phase solution in a volume ratio of 1 (0.2-50), wherein the volume ratio is preferably 1: (0.5-20). Under the existence of a surfactant, a large amount of submicron cavitation bubbles escape, and are encapsulated by lipid nanoparticles together with nucleic acid to obtain a crude product;
s4, post-treatment of products
First, a buffer solution is added to the crude product obtained in step S3 for dilution. The diluted crude product is then subjected to solvent displacement and concentration using tangential flow techniques. And finally, carrying out aseptic treatment on the concentrated product to obtain a final product.
In a preferred embodiment, the process for preparing the nucleic acid vaccine or pharmaceutical composition of the present invention comprises the steps of:
s1, solution configuration
(1) Dissolving ionizable cations, neutral phospholipids, cholesterol, PEG phospholipids and surfactant in an organic solvent to obtain an organic phase;
(2) Dispersing nucleic acid into an acidic buffer solution with ph=3-6 as an aqueous phase; wherein the acidic buffer solution comprises one or two of acetic acid-sodium acetate buffer solution and citrate buffer solution;
s2, preparation of gas-rich solution
And (3) purging the water phase and the organic phase for 0.5-2 hours by using gas to obtain gas-rich solution, namely gas-rich water phase solution and gas-rich organic phase solution respectively.
S3, mixing the solutions
Mixing the gas-rich aqueous phase solution and the gas-rich organic phase solution according to the volume ratio of 1 (0.2-50) by utilizing an ultrasonic-assisted micro-fluidic technology, wherein the preferred volume ratio is 1: (0.5-20). Under the existence of a surfactant, a large amount of submicron cavitation bubbles escape, and are encapsulated by lipid nanoparticles together with nucleic acid to obtain a crude product;
s4, post-treatment of products
First, a buffer solution is added to the crude product obtained in step S3 for dilution. The diluted crude product is then subjected to solvent displacement and concentration using tangential flow techniques. And finally, carrying out aseptic treatment on the concentrated product to obtain a final product.
In a preferred experimental scheme, the preparation process of the small molecule chemical drug combination comprises the following steps:
s1, solution configuration
(1) Dissolving ionizable cation and/or permanent cation lipid, neutral phospholipid, cholesterol, PEG phospholipid, and surfactant in organic solvent to obtain organic phase;
(2) Dissolving doxorubicin in water, and adjusting pH to 7.4 with phosphate buffer solution to obtain water phase;
s2, preparation of gas-rich solution
And (3) purging the water phase and the organic phase for 0.5-2 hours by using gas to obtain gas-rich solution, namely gas-rich water phase solution and gas-rich organic phase solution respectively.
S3, mixing the solutions
Under the action of ultrasound, mixing the gas-rich aqueous phase solution and the gas-rich organic phase solution in a volume ratio of 1 (0.2-50). Under the existence of a surfactant, a large amount of submicron cavitation bubbles escape, and are encapsulated by lipid nano particles together with doxorubicin, so that a crude product is obtained;
s4, post-treatment of products
First, a buffer solution is added to the crude product obtained in step S3 for dilution. The diluted crude product is then subjected to solvent displacement and concentration using tangential flow techniques. And finally, carrying out aseptic treatment on the concentrated product to obtain a final product.
In a preferred experimental scheme, the preparation process of the small molecule chemical drug combination comprises the following steps:
s1, solution configuration
(1) Dissolving ionizable cations, neutral phospholipids, cholesterol, PEG phospholipids, surfactants, and vitamin a in an organic solvent as an organic phase;
(2) Phosphate buffer salt solution (ph=7.4) was used as the aqueous phase;
s2, preparation of gas-rich solution
And (3) purging the water phase and the organic phase for 0.5-2 hours by using gas to obtain gas-rich solution, namely gas-rich water phase solution and gas-rich organic phase solution respectively.
S3, mixing the solutions
Under the action of ultrasound, mixing the gas-rich aqueous phase solution and the gas-rich organic phase solution in a volume ratio of 1 (0.2-50). Under the existence of a surfactant, a large amount of submicron cavitation bubbles escape and are encapsulated by lipid nano particles together with vitamin A to obtain a crude product;
s4, post-treatment of products
First, a buffer solution is added to the crude product obtained in step S3 for dilution. The diluted crude product is then subjected to solvent displacement and concentration using tangential flow techniques. And finally, carrying out aseptic treatment on the concentrated product to obtain a final product.
The present invention provides a novel lipid nanoparticle having a structure different from that of conventional lipid nanoparticles. The novel lipid nanoparticle consists of ionizable cationic lipid and/or permanent cationic lipid, neutral auxiliary phospholipid, steroid compound, PEG phospholipid, gas and water, has a multi-vesicle structure, and consists 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 of nucleic acid or micromolecular chemical medicaments, and a corresponding vaccine or pharmaceutical composition is prepared by rapidly mixing raw materials under the conditions of ultrasound and the existence of a surfactant, and the principle is as follows: under the action of ultrasonic wave and in the presence of surfactant, a large quantity of submicron-order bubbles are produced. The self-assembly process of the ionizable cationic lipid, neutral auxiliary phospholipid, cholesterol and PEG phospholipid is enhanced, and simultaneously submicron cavitation bubbles and water are wrapped by a phospholipid membrane to form a multi-vesicle structure. In the process of cell uptake, the novel lipid nanoparticle can provide an environment with forward biological effects such as oxygen enrichment or hydrogen enrichment and the like while delivering nucleic acid, and the nucleic acid delivery efficiency is remarkably improved. The novel lipid nanoparticle can also be used for delivering small molecule chemicals to improve the bioavailability of the small molecule chemicals.
Compared with the prior art, the invention has the following beneficial effects:
(1) The multi-vesicle lipid nanoparticle formed by the lipid vesicle with the inner cavity being water and the lipid vesicle with the inner cavity being gas can provide an environment with positive biological effects such as oxygen enrichment or hydrogen enrichment and the like while delivering nucleic acid or small molecular chemical drugs, and the delivery efficiency of the nucleic acid or the small molecular chemical drugs is obviously improved.
(2) The lipid vesicle with the inner cavity being gas can be used as an artificial cavitation nucleus, and can generate cavitation effect under ultrasonic radiation to expand, compress or burst, so that the permeability of cell membranes and vascular systems is enhanced. In addition, the jet generated by cavitation releases energy within the cell, and the cell membrane will instantaneously open "acoustic holes" which are good channels for the entry of pharmaceutically active components into the cell. Therefore, in the process of taking the lipid nanoparticle provided by the invention by cells, the directional blasting of the air sac-containing bubbles can be realized by applying ultrasound in vitro, and the bioavailability and targeting of nucleic acid or small molecule chemical drugs are expected to be further improved.
Drawings
FIG. 1, a cryoelectron micrograph of mRNA-lipid nanoparticles in example 1;
FIG. 2, mRNA-lipid nanoparticle dynamic light scattering particle size distribution diagram in example 1;
FIG. 3, results of statistics of fluorescent microscopy of lipid nanoparticle EYFP mRNA (mRNA of intense yellow fluorescent protein) in example 1;
FIG. 4 is a view of in vivo imaging of the delivery of Luc-mRNA in lipid nanoparticles of example 1.
FIG. 5, a cryoelectron micrograph of mRNA-lipid nanoparticles of comparative example 1;
FIG. 6, mRNA-lipid nanoparticle dynamic light scattering particle size distribution diagram of comparative example 1;
FIG. 7 is a view of in vivo imaging of the delivery of Luc-mRNA in lipid nanoparticles of comparative example 1.
Detailed Description
In order that those skilled in the art will better understand the present invention, a more particular description of the invention will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings, wherein it is to be understood that the illustrated embodiments are merely exemplary of some, but not all, of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, shall fall within the scope of the present invention. It should be noted that, without conflict, the embodiments of the present invention and features of the embodiments may be combined with each other. The present invention will be described in detail with reference to examples.
Example 1
(1) Preparation of mRNA-lipid nanoparticles by ultrasound-assisted microfluidic technology
Firstly, (2, 3-dioleoyl-propyl) -trimethylamine (SM 102) is selected as ionizable cationic lipid, 1, 2-distearoyl-sn-glycerophosphorylcholine (DSPC) is selected as neutral auxiliary phospholipid, cholesterol is selected as steroid, 1, 2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000 (DMG-PEG 2000) is selected as PEG phospholipid, and the mole percentages of SM102, DSPC, cholesterol and DMG-PEG2000 are respectively 50%, 10%, 38.5% and 1.5%. The lipid substances and Tween 80 are simultaneously dissolved in absolute ethyl alcohol to be used as organic phase solution, the total concentration of the lipid substances is 20mg/mL, and the mass fraction of the Tween 80 is 0.1%. For in vitro cell transfection experiments, the aqueous phase solution was prepared as follows: EYFP mRNA (mRNA of the intense yellow fluorescent protein) was dispersed in citrate buffer (10 mM, pH=4.0) with a molar ratio of EYFP mRNA to lipid controlled to be 1:15; for in vivo transfection experiments, the aqueous phase solution was prepared as follows: luc-mRNA (firefly luciferase mRNA) was dispersed in citrate buffer (10 mm, ph=4.0) and the molar ratio of Luc-mRNA to lipid was controlled 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 phase solution and the aqueous phase solution are rapidly mixed by utilizing an ultrasonic-assisted microfluidic technology at room temperature, the diameter of a reactor channel is 1 mm, the volume flow ratio of the organic phase solution to the aqueous phase solution is 3:1, the total volume flow is 8mL/min, the ultrasonic power is 10W, the ultrasonic frequency is 20KHz, and the ultrasonic treatment time is 0.8s. The resulting mRNA-lipid nanoparticle solution was rapidly transferred to a 50mL ultrafiltration tube and the resulting solution was diluted 10-fold with 1 XPBS 10 buffer. Subsequently, the mRNA-lipid nanoparticle solution after ultrafiltration was collected by centrifugation at room temperature for 15min, and the volume was fixed to a concentration of 160ug/mL with 1 XPBS buffer. And finally, filtering the sample by adopting a 0.22 mu m polyether sulfone filter membrane, and performing aseptic treatment to obtain a final product. Serial labeling of Luc-mRNA-lipid nanoparticles: the morphology of the prepared mRNA-lipid nanoparticles was characterized using cryoelectron microscopy, and the results are shown in fig. 1. The lipid nano-particles prepared by the frozen electron microscope photo are in a multi-vesicle shape and are composed 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, and the result is shown in FIG. 2, the particle size of the prepared mRNA-lipid nanoparticles is 98nm, and the polydispersity is as low as 0.05.mRNA encapsulation efficiency was determined by RiboGreen (Thermofisher) and was 95%.
(2) In vitro cell transfection
And (3) detecting the in vitro delivery efficiency of the EYFP-mRNA-lipid nanoparticle prepared in the step (1) on a cell model. Using a549 cells, 96-well plates were plated at a cell density of 30000 cells/well. After 18 hours, in vitro cell transfection experiments were performed with 50ng/mL EYFP-mRNA-lipid nanoparticles, with five duplicate wells per set of experiments. Transfection efficiency was measured after 24 hours using flow cytometry and fluorescence microscopy (see figure 3). In vitro cell transfection experiment results show that the transfection efficiency of EYFP-mRNA reaches 99.38%, and the fluorescence intensity is more than 10 6 . The lipid nanoparticle provided by the invention has high delivery efficiency on EYFP-mRNA, and is an excellent mRNA delivery carrier.
(3) In vivo transfection experiments
The Luc-mRNA-lipid nanoparticles prepared in step (1) were tested for delivery efficiency in a mouse animal model by injecting Luc-mRNA-lipid nanoparticles into mice (BALB/c, 8 weeks old) by intravenous and intramuscular injection, respectively, at a dose of 10 μg per mouse and three mice per group. After 6 hours, mice were injected with a luciferin substrate D-luciferin and subjected to in vivo imaging using a in vivo imager, the luminous intensity of the mice was measured, and the in vivo delivery efficiency of nanoliposome particles was examined (see fig. 4). According to the in vivo delivery efficiency results, the nano liposome particles provided by the invention can well deliver Luc-mRNA, and have the advantages of high delivery efficiency, good survival state of mice and good safety.
Example 2
Firstly, (2, 3-dioleoyl-propyl) -trimethylamine (SM 102) is selected as ionizable cationic lipid, 1, 2-distearoyl-sn-glycerophosphorylcholine (DSPC) is selected as neutral auxiliary phospholipid, cholesterol is selected as steroid, 1, 2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000 (DMG-PEG 2000) is selected as PEG phospholipid, and the mole percentages of SM102, DSPC, cholesterol and DMG-PEG2000 are respectively 40%, 20%, 38.5% and 1.5%. The lipid substances and Tween 80 are simultaneously dissolved in absolute ethyl alcohol to be used as organic phase solution, the total concentration of the lipid substances is 40mg/mL, and the mass fraction of the Tween 80 is 1%. The aqueous solution was prepared by dispersing EYFP mRNA (mRNA of the intense yellow fluorescent protein) in citrate buffer (10 mm, ph=4.0) and controlling the molar ratio of EYFP mRNA to lipid material to be 1:15. Subsequently, the aqueous and organic phases were purged with sterile hydrogen for 2 hours to obtain an oxygen-enriched gas solution. At room temperature, the organic phase solution and the aqueous phase solution are rapidly mixed by utilizing an ultrasonic-assisted microfluidic technology, the diameter of a reactor channel is 2 millimeters, the volume flow ratio of the organic phase solution to the aqueous phase solution is 3:1, the total volume flow is 16mL/min, the ultrasonic power is 20W, the ultrasonic frequency is 28KHz, and the ultrasonic treatment time is 1.2s. The resulting mRNA-lipid nanoparticle solution was rapidly transferred to a 50mL ultrafiltration tube and the resulting solution was diluted 10-fold with 1 XPBS 10 buffer. Subsequently, the mRNA-lipid nanoparticle solution after ultrafiltration was collected by centrifugation at room temperature for 15min, and the volume was fixed to a concentration of 160ug/mL with 1 XPBS buffer. Finally, the sample is filtered by a 0.22 mu m polyethersulfone filter membrane and subjected to aseptic treatment. The lipid nano-particles prepared by the frozen electron microscope photo are in a multi-vesicle shape and are composed 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 are characterized by a dynamic light scattering method, the particle size of the prepared mRNA-lipid nanoparticles is 82nm, and the polydispersity coefficient is as low as 0.06.mRNA encapsulation efficiency was measured by RiboGreen (Thermofisher), and was 93%
Example 3
Firstly, (2, 3-dioleoyl-propyl) -trimethylamine (SM 102) is selected as ionizable cationic lipid, 1, 2-distearoyl-sn-glycerophosphorylcholine (DSPC) is selected as neutral auxiliary phospholipid, cholesterol is selected as steroid, 1, 2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000 (DMG-PEG 2000) is selected as PEG phospholipid, and the mole percentages of SM102, DSPC, cholesterol and DMG-PEG2000 are 40%, 10%, 49% and 1%, respectively. The lipid substances and Tween 80 are simultaneously dissolved in absolute ethyl alcohol to be used as organic phase solution, the total concentration of the lipid substances is 20mg/mL, and the mass fraction of the Tween 80 is 0.5%. The aqueous solution was prepared by dispersing EYFP mRNA (mRNA of the intense yellow fluorescent protein) in citrate buffer (10 mm, ph=4.0) and controlling the molar ratio of EYFP mRNA to lipid material to be 1:12. Subsequently, the aqueous and organic phases were purged with sterile oxygen for 0.5 hours to obtain an oxygen-enriched gas solution. At room temperature, the organic phase solution and the aqueous phase solution are rapidly mixed by utilizing an ultrasonic-assisted microfluidic technology, the diameter of a reactor channel is 1 mm, the volume flow ratio of the organic phase solution to the aqueous phase solution is 3:1, the total volume flow is 16mL/min, the ultrasonic power is 30W, the ultrasonic frequency is 40KHz, and the ultrasonic treatment time is 0.5s. The resulting mRNA-lipid nanoparticle solution was rapidly transferred to a 50mL ultrafiltration tube and the resulting solution was diluted 10-fold with 1 XPBS 10 buffer. Subsequently, the mRNA-lipid nanoparticle solution after ultrafiltration was collected by centrifugation at room temperature for 15min, and the volume was fixed to a concentration of 160ug/mL with 1 XPBS buffer. Finally, the sample is filtered by a 0.22 mu m polyethersulfone filter membrane and subjected to aseptic treatment. The lipid nano-particles prepared by the frozen electron microscope photo are in a multi-vesicle shape and are composed 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 are characterized by a dynamic light scattering method, the particle size of the prepared mRNA-lipid nanoparticles is 75nm, and the polydispersity coefficient is as low as 0.05.mRNA encapsulation efficiency was determined by RiboGreen (Thermofisher), and was 94%
Example 4
Firstly, 4- (N, N-dimethylamino) butanoic acid (diimine) methyl ester (Dlin-MC 3-DMA) is selected as ionizable cationic lipid, 1, 2-distearoyl-sn-glycerophosphorylcholine (DSPC) is selected as neutral auxiliary phospholipid, cholesterol is selected as steroid, 1, 2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000 (DMG-PEG 2000) is selected as PEG phospholipid, and the mole percentages of SM102, DSPC, cholesterol and DMG-PEG2000 are respectively 60%, 20%, 29% and 1%. The lipid substances and Tween 80 are simultaneously dissolved in absolute ethyl alcohol to be used as organic phase solution, the total concentration of the lipid substances is 20mg/mL, and the mass fraction of the Tween 80 is 0.1%. The aqueous solution was prepared by dispersing EYFP mRNA (mRNA of the intense yellow fluorescent protein) in citrate buffer (10 mm, ph=4.0) and controlling the molar ratio of EYFP mRNA to lipid material to be 1:15. Subsequently, the aqueous and organic phases were 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 rapidly mixed by utilizing an ultrasonic-assisted microfluidic technology, the volume flow ratio of the organic phase solution to the aqueous phase solution is 3:1, the total volume flow is 16mL/min, the ultrasonic power is 10W, the ultrasonic frequency is 20KHz, and the ultrasonic treatment time is 1.2s. The resulting mRNA-lipid nanoparticle solution was rapidly transferred to a 50mL ultrafiltration tube and the resulting solution was diluted 10-fold with 1 XPBS 10 buffer. Subsequently, the mRNA-lipid nanoparticle solution after ultrafiltration was collected by centrifugation at room temperature for 15min, and the volume was fixed to a concentration of 160ug/mL with 1 XPBS buffer. Finally, the sample is filtered by a 0.22 mu m polyethersulfone filter membrane and subjected to aseptic treatment. The lipid nano-particles prepared by the frozen electron microscope photo are in a multi-vesicle shape and are composed 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 are characterized by a dynamic light scattering method, the particle size of the prepared mRNA-lipid nanoparticles is 90nm, and the polydispersity coefficient is as low as 0.05.mRNA encapsulation efficiency was determined by RiboGreen (Thermofisher), and was 96%
Example 5
Firstly, 4- (N, N-dimethylamino) butanoic acid (diimine) methyl ester (Dlin-MC 3-DMA) is selected as ionizable cationic lipid, 1, 2-distearoyl-sn-glycerophosphorylcholine (DSPC) is selected as neutral auxiliary phospholipid, cholesterol is selected as steroid, 1, 2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000 (DMG-PEG 2000) is selected as PEG phospholipid, and the mole percentages of SM102, DSPC, cholesterol and DMG-PEG2000 are respectively 60%, 20%, 29% and 1%. The lipid substances, tween 80 and vitamin D are simultaneously dissolved in absolute ethyl alcohol to be used as organic phase solution, wherein the total concentration of the lipid substances is 20mg/mL, the mass fraction of Tween is 0.1%, and the concentration of the vitamin D is 100mg/mL. The aqueous phase was citrate buffer (10 mm, ph=4.0). Subsequently, the aqueous and organic phases were purged with sterile oxygen for 0.5 hours to obtain an oxygen-enriched solution. At room temperature, the organic phase solution and the aqueous phase solution are rapidly mixed by utilizing an ultrasonic-assisted microfluidic technology, the volume flow ratio of the organic phase solution to the aqueous phase solution is 3:1, the total volume flow is 20mL/min, the ultrasonic power is 10W, the ultrasonic frequency is 20KHz, and the ultrasonic treatment time is 0.3s. The resulting vitamin D-lipid nanoparticle solution was rapidly transferred to a 50mL ultrafiltration tube and the resulting solution was diluted 10-fold with 1 x PBS10 buffer solution. Subsequently, the vitamin D-lipid nanoparticle solution after ultrafiltration was collected by centrifugation at room temperature for 15min, and the volume was fixed to a concentration of 160ug/mL with 1 XPBS buffer solution. Finally, the sample is filtered by a 0.22 mu m polyethersulfone filter membrane and subjected to aseptic treatment. The lipid nano-particles prepared by the frozen electron microscope photo are in a multi-vesicle shape and are composed 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 are characterized by utilizing a dynamic light scattering method, the particle size of the prepared vitamin D-lipid nanoparticles is 75nm, and the polydispersity coefficient is as low as 0.05.
Comparative example 1
(1) mRNA-lipid nanoparticle preparation by microfluidic technology
Firstly, (2, 3-dioleoyl-propyl) -trimethylamine (SM 102) is selected as ionizable cationic lipid, 1, 2-distearoyl-sn-glycerophosphorylcholine (DSPC) is selected as neutral auxiliary phospholipid, cholesterol is selected as steroid, 1, 2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000 (DMG-PEG 2000) is selected as PEG phospholipid, and the mole percentages of SM102, DSPC, cholesterol and DMG-PEG2000 are respectively 50%, 10%, 38.5% and 1.5%. The lipid substance and tween 80 are simultaneously dissolved in absolute ethyl alcohol to be used as organic phase solution, the total concentration of the lipid substance is 20mg/mL, and the mass fraction of tween 80 is 0.1%. The preparation process of the aqueous phase solution comprises the following steps: luc-mRNA (firefly luciferase mRNA) was dispersed in citrate buffer (10 mm, ph=4.0) and the molar ratio of Luc-mRNA to lipid was controlled 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 phase solution and the aqueous phase solution were rapidly mixed at room temperature using microfluidic technology, the reactor channel geometry and configuration were the same as in example 1, the volume flow ratio of the organic phase solution to the aqueous phase solution was 3:1, and the total volume flow was 8mL/min. The resulting mRNA-lipid nanoparticle solution was rapidly transferred to a 50mL ultrafiltration tube and the resulting solution was diluted 10-fold with 1 XPBS 10 buffer. Subsequently, the mRNA-lipid nanoparticle solution after ultrafiltration was collected by centrifugation at room temperature for 15min, and the volume was fixed to a concentration of 160ug/mL with 1 XPBS buffer. And finally, filtering the sample by adopting a 0.22 mu m polyether sulfone filter membrane, and performing aseptic treatment to obtain a final product. The morphology of the prepared mRNA-lipid nanoparticles was characterized using cryoelectron microscopy, and the results are shown in fig. 5. The frozen electron microscope photo shows that the prepared lipid nanoparticle has a single vesicle shape, and the inner cavity is water. The average particle size and distribution of the prepared mRNA-lipid nanoparticles were characterized by dynamic light scattering, and the result is shown in FIG. 6, wherein the particle size of the prepared mRNA-lipid nanoparticles is 115nm, and the polydispersity is 0.12.mRNA encapsulation efficiency was determined by RiboGreen (Thermofisher) and was 85%.
(2) In vivo transfection experiments
The Luc-mRNA-lipid nanoparticles prepared in step (1) were tested for delivery efficiency in a mouse animal model by injecting Luc-mRNA-lipid nanoparticles into mice (BALB/c, 8 weeks old) by intravenous and intramuscular injection, respectively, at a dose of 10 μg per mouse and three mice per group. After 6 hours, mice were injected with a luciferin substrate D-luciferin and subjected to in vivo imaging using a in vivo imager, the luminous intensity of the mice was measured, and the in vivo delivery efficiency of nanoliposome particles was examined (see fig. 7). As can be seen, the in vivo delivery efficiency of single vesicle-like and gas-free lipid nanoparticles was significantly lower than that of the gas-containing multi-vesicle-like lipid nanoparticles prepared in example 1.

Claims (10)

1. The gas-containing multivesicular lipid nanoparticle is characterized by comprising ionizable cationic lipid and/or permanent cationic lipid, neutral auxiliary phospholipid, steroid, polyethylene glycol phospholipid, gas and water; the structure is a multi-vesicle, and the multi-vesicle is composed of a lipid vesicle with a water inner cavity and a lipid vesicle with a gas inner cavity; wherein, the lipid vesicle with the inner cavity of water is positioned in the center of the multi-vesicle, and the lipid vesicle with the inner cavity of gas is distributed at the edge of the lipid vesicle with the inner cavity of water.
2. The lipid nanoparticle of claim 1, consisting of one lipid vesicle with water in its lumen and one or more lipid vesicles with gas in its lumen distributed at its edges.
3. The lipid nanoparticle according to claim 1, wherein the ionizable and/or permanent cationic lipid is one or more of 4- (N, N-dimethylamino) butanoic acid (diiodol) methyl ester, 2-diiodol-4- (2-dimethylaminoethyl) -1, 3-dioxan, 2, 3-dioleyloxypropyl-1-bromotrimethylamine, (2, 3-dioleoyl-propyl) -trimethylamine, heptadecan-9-yl-8- ((2-hydroxyethyl) (6-oxo-6- ((decyloxy) hexyl) amino) octanoate), ((4-hydroxybutyl) azadiyl) bis (hexane-6, 1-diyl) bis (2-hexyl decanoate), preferably 4- (N, N-dimethylamino) butanoic acid (diiodol) methyl ester, heptadecan-9-yl-8- ((2-hydroxyethyl) (6-oxo-6- ((decyloxy) hexyl) amino) and 4-hydroxybutyl) bis (2-hexyl) decanoate;
the neutral auxiliary phospholipid is one or more of 1, 2-distearoyl-sn-glycerophosphorylcholine, dioleoyl phosphatidylcholine, 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine, dipalmitoyl phosphatidylcholine, dimyristoyl phosphatidylcholine or (2, 3-dioleoyl-propyl) -trimethylamine sulfate, preferably one or more of 1, 2-distearoyl-sn-glycerophosphorylcholine, 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine;
the steroid compound is one or more of cholesterol, sitosterol, rock soap sterol, campesterol and stigmasterol, preferably cholesterol;
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 ditetradecylacetamide, preferably 1, 2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol 2000;
the gas is one or more of oxygen, hydrogen and nitrogen.
4. Lipid nanoparticle according to claim 1, wherein the mole percentage of ionizable and/or permanent cationic lipids is 40-70%, preferably 45-60%, without water and gas;
the mole percentage of the neutral auxiliary phospholipid is 2-25%, preferably 5-20% when water and gas are not counted; the molar percentage of said steroid, excluding water and gas, is 20% to 60%, preferably 30% to 50%;
the polyethylene glycol phospholipid is present in a molar percentage of 0.25% to 5%, preferably 0.8% to 2%, when water and gas are not counted.
5. The lipid nanoparticle according to claim 1, wherein the water comprises 20-60%, preferably 30-55% by volume of the lipid nanoparticle;
the gas comprises 2% -20%, preferably 5% -15% of the volume fraction of the lipid nanoparticle.
6. Lipid nanoparticle according to claim 1, characterized in that the average particle size is 50-250nm, preferably 60-150nm.
7. A vaccine or pharmaceutical composition comprising the lipid nanoparticle of any one of claims 1-5 and a pharmaceutically active component.
8. The vaccine or pharmaceutical composition according to claim 7, wherein the pharmaceutically active component is a nucleic acid or a small molecule chemical;
preferably, the nucleic acid is a small interfering RNA or a messenger RNA; the small molecular chemical medicine comprises one or more of doxorubicin, curcumin, taxol, docetaxel, carotenoid, vitamin A, vitamin C and vitamin D;
preferably, the messenger RNA is a linear non-self-replicating messenger RNA, a linear self-replicating messenger RNA, or a circular messenger RNA.
9. The vaccine or pharmaceutical composition according to claim 7, wherein the preparation process comprises the steps of:
s1, solution configuration
(1) Dissolving ionizable cation and/or permanent cation lipid, neutral auxiliary phospholipid, steroid, polyethylene glycol phospholipid, and surfactant in organic solvent to obtain organic phase;
(2) When the pharmaceutically active component is a nucleic acid, dispersing the nucleic acid into an acidic buffer solution having ph=3-6 as an aqueous phase;
(3) When the active pharmaceutical ingredient is a small molecular chemical, if the small molecular chemical is soluble in water, the small molecular chemical is preferentially dissolved in water, and the pH value of the water phase is controlled to be 3-7 by using a buffer solution to serve as the water phase; if the micromolecular chemical medicine is insoluble in water, dissolving the micromolecular chemical medicine, ionizable cations and/or permanent cationic lipid, neutral auxiliary phospholipid, steroid compound and polyethylene glycol phospholipid in an organic solvent at the same time, taking pure water as a water phase, and controlling the pH value of the water phase to be 3-7 by utilizing a buffer solution;
s2, preparation of gas-rich water phase and organic phase
The aqueous phase and the organic phase were purged with gas for 0.5 to 2 hours to obtain a gas-rich aqueous phase and an organic phase, respectively.
S3, mixing the solutions
Under the action of ultrasound, rapidly mixing a gas-rich organic phase and a gas-rich water phase in a predetermined volume ratio, and forming a large number of submicron cavitation bubbles in the presence of a surfactant, wherein the bubbles and the active components of the drug are encapsulated by lipid nanoparticles to obtain a crude product;
s4, post-treatment of products
Firstly, adding a buffer solution into the crude product obtained in the step S3 for dilution; then, the diluted crude product is subjected to solvent replacement and concentration by using a tangential flow technology; and finally, carrying out aseptic treatment on the concentrated product to obtain a final product.
10. The vaccine or pharmaceutical composition according to claim 7, wherein in step S1, the organic solvent is one or more of ethanol, acetonitrile, acetone, tetrahydrofuran, N-dimethylformamide; the total concentration of the sum of the ionizable and/or permanent cationic lipid, neutral helper phospholipid, steroid, PEG phospholipid in the organic phase is 0.5-40mg/mL, preferably 5-20mg/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%;
in step S2, when the pharmaceutically active component is a nucleic acid, the acidic buffer solution includes one or two of acetic acid-sodium acetate buffer solution and citrate buffer solution; the concentration of the nucleic acid in the aqueous phase is 0.02-5mg/mL, preferably 0.1-1mg/mL; when the active pharmaceutical ingredient is a small molecular chemical drug, the buffer solution comprises one or two of acetic acid-sodium acetate buffer solution, citrate buffer solution and phosphate buffer solution; the concentration of the small molecule chemical in the water phase or the organic phase is 0.02-300mg/mL, preferably 0.1-180mg/mL;
in the step S3, the ultrasonic power is 1-1000W, preferably 5-200W; the ultrasonic frequency is 20KHz-1MHz, preferably 20-100KHz; the sonication time is from 0.01 seconds to 1 hour, preferably from 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);
in step S4, the buffer solution is a phosphate buffer solution.
CN202310853746.6A 2023-07-12 2023-07-12 Gas-containing multivesicular lipid nanoparticle, application and preparation method thereof Pending CN116807997A (en)

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