CN115154439B - mRNA lipid nanoparticle delivery system and preparation method and application thereof - Google Patents
mRNA lipid nanoparticle delivery system and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses an mRNA lipid nanoparticle delivery system, and a preparation method and application thereof. A delivery system of mRNA drug is lipid nanoparticle loaded with one or more mRNA. The lipid nanoparticles are prepared from ionizable cationic lipid DHA-1, cholesterol and phospholipid polyethylene glycol derivative DMG-PEG2000 as raw materials. The lipid component of the invention is simple, only three components are needed, auxiliary phospholipid is not needed, the cost is lower, the process is simpler and more convenient, and the lipid component is suitable for large-scale production. The invention firstly applies for patents by taking DHA1 which is a cationic lipid as the main component of the nucleic acid delivery dosage form, and the raw materials are not limited by patent barriers. Animal experiments prove that the delivery system has the actual effect reaching the effect of the nucleic acid delivery system adopted by the latest international products on the market and has the value of commercial production.
Description
Technical Field
The invention belongs to the field of medical biology, and particularly relates to a non-viral vector-based mRNA nucleic acid drug intracellular delivery system, a preparation method and application, which can be produced and applied in a large scale.
Background
It is well known that new coronary epidemics greatly advance the development of mRNA vaccines. Compared with the conventional vaccine, the mRNA vaccine has the advantages of low cost, high production efficiency and high safety, and has the potential of synthesizing any protein. Therefore, the method has great application potential for novel infectious viruses which cannot be responded by the traditional vaccine. However, mRNA vaccines have been limited in their use due to their instability, susceptibility to degradation by rnases, and low in vivo delivery efficiency. To achieve widespread use of mRNA vaccines, delivery techniques need to be addressed with emphasis. The mRNA vaccine needs a suitable delivery vehicle to deliver it into the body for better immunization, so the development of an efficient and nontoxic delivery system is the key to the success of the mRNA vaccine.
In recent years, nanotechnology has been developed rapidly, and its application in the biomedical field has also been attracting much attention. Nano delivery systems (nanoparticularly delivery systems) refer to drug delivery systems with particle diameters on the nano scale (1-1000 nm), which are mainly used for concentrating and loading drugs in various forms such as embedding, adsorption, encapsulation or covalent bonding, and the like, so as to deliver the drugs to specific organs or cells in a targeted manner. Lipid Nanoparticles (LNPs) were developed from Stabilized plasmid-Lipid particles (SPLP). Lipid nanoparticles are generally prepared from at least raw materials comprising ionizable cationic lipids, phospholipid helper lipids, cholesterol, and phospholipid polyethylene glycol derivatives. In order to improve transfection efficiency, the skilled person has made many optimizations and innovations in the selection of the above substances, such as CN 202010225030.8, which increases mRNA transfection efficiency by selecting MVL5 as an ionizable cationic lipid.
Disclosure of Invention
The present invention aims to overcome the above defects of the prior art and provide a lipid nanoparticle and application thereof.
The invention also aims to provide an mRNA drug delivery system prepared based on the lipid nanoparticle and application thereof.
It is a further object of the invention to provide a method for preparing the delivery system.
The purpose of the invention can be realized by the following technical scheme:
a lipid nanoparticle is prepared from an ionizable cationic lipid DHA-1, cholesterol and a phospholipid polyethylene glycol derivative DMG-PEG 2000.
The ionizable cationic lipid DHA-1 has the following structural formula:
the phospholipid polyethylene glycol derivative DMG-PEG2000 (CAS No.: 160743-62-4) has the following structure:
in a preferred embodiment of the present invention, the cholesterol is selected from one or both of animal-derived cholesterol and plant-derived cholesterol, and is preferably plant-derived cholesterol.
Preferably, the molar ratio of the ionizable cationic lipid to the plant-derived cholesterol to the phospholipid polyethylene glycol derivative is (25-60): (25-70): (1-5), more preferably (45-55): (35-40): (1-5).
In a preferred embodiment of the present invention, the lipid nanoparticle has an average particle size of 60 to 140nm, preferably 80 to 120nm.
The lipid nanoparticle disclosed by the invention is applied to preparation of a delivery system of an mRNA (messenger ribonucleic acid) drug.
An mRNA drug delivery system is a lipid nanoparticle of the present invention loaded with one or more mrnas.
As a preferred aspect of the present invention, the mRNA drug delivery system is prepared by a microfluidic device.
Preferably, the lipid nanoparticles have an average particle size of 60-140nm, preferably 80-120nm; under neutral environmental conditions, mRNA-LNP has a Zeta potential of-15 mV to +5mV.
A method of making the mRNA drug delivery system of the present invention, comprising the steps of:
s1, preparing an mRNA solution as a water phase;
s2, preparing an absolute ethyl alcohol solution containing all components of the lipid nanoparticles as an organic phase, wherein the molar ratio of the ionizable cationic lipid to the plant-derived cholesterol to the phospholipid polyethylene glycol derivative is (25-60): (25-70): (1-5), preferably (45-55): (35-40): (1-5), the nitrogen-phosphorus ratio of the ionizable cationic lipid DHA-1 to the mRNA is (3-8): 1;
and S3, controlling the flow rate ratio of the aqueous phase to the organic phase and the total flow rate of the mixing pipeline by adopting a microfluidic method, mixing the two solutions in a microfluidic chip, and combining positively charged cationic lipid with negatively charged mRNA to prepare the mRNA lipid nanoparticles.
In a preferred embodiment of the present invention, in S1, the mRNA solution is prepared by dissolving mRNA in a buffer solution, wherein the concentration of the mRNA is 0.01-100mM, preferably 1-10mM; the buffer is selected from 5-10mM citric acid buffer with pH3.0, preferably 5mM citric acid buffer with pH 3.0.
In a preferred embodiment of the present invention, in S2, the ratio of nitrogen to phosphorus (N: P) of cationic lipid to mRNA is 5:1 preparing a lipid mixed solution as an organic phase.
As one preferable aspect of the present invention, in S3, the flow rate ratio of the aqueous phase to the organic phase is 3: 1; the total flow rate of the mixing line of the aqueous phase and the organic phase was 4ml/min.
Preferably, the preparation method further comprises purifying the S4, mRNA-lipid nanoparticles by ultrafiltration with an ultrafiltration tube; the size of the interception aperture is 100kd; the mRNA-LNP concentration conditions were 30 ℃ fixed angle rotor, 2000g,25 ℃ room temperature centrifugation.
The delivery system of the invention is applied to the preparation of mRNA drugs or vaccines, and the mRNA is preferably used for the complete mRNA molecules of functional proteins, therapeutic monoclonal antibodies, B cell epitopes, T cell epitopes or tumor neoantigen peptide segments expressed by preventing or treating diseases.
Has the advantages that:
1. the delivery system of the present invention consists of only three components, respectively cationic lipid, plant-derived cholesterol and phospholipid polyethylene glycol derivatives. The delivery system of the nucleic acid drugs on the market and most of the patents of the delivery system comprise four components, namely cationic lipid, auxiliary phospholipid, animal-derived cholesterol and phospholipid polyethylene glycol derivatives.
2. The invention adopts the DHA-1 cationic lipid as the main component of the nucleic acid delivery dosage form for the first time, the cationic lipid DHA-1 is structurally combined with the advantage design of the lipid of the vaccine on the market abroad, the side chain of the tertiary amine is optimized and modified, and the delivery effect and the safety are high.
3. The plant source cholesterol is a plant source, and the safety is better than that of a conventional animal source.
4. The phospholipid polyethylene glycol derivative is DMG-PEG2000, and compared with the conventional DSPE-PEG2000, the phospholipid polyethylene glycol derivative has higher delivery efficiency, and specifically comprises the following components: the modified fatty acid chain of DMG-PEG2000 is myristic acid C14 which is much shorter than the stearic acid C18 chain of common DSPE-PEG2000, so that the most direct result is that the lipid 'anchor' is embedded into a lipid membrane to be shallow, and is easy to fall off in the systemic circulation process, and the uptake of target cells to the liposome is improved; in addition, the short chain (myristic acid, C14) of DMG-PEG2000 has a shorter half-life period, is degraded more quickly than the long chain (stearic acid, C18), and has better safety.
5. The nucleic acid delivery formulations are prepared in an advanced microfluidic manner suitable for scale-up production.
Drawings
FIG. 1 is a schematic diagram of the preparation process of the present invention.
FIG. 2 shows the results of imaging of mRNA-LNP of the present invention and control formulations including the Modena neo-corona vaccine formulation mRNA-LNP after 6h intramuscular injection into the legs of mice, mouse numbers L1-L3.
FIG. 3 shows the results of imaging the mRNA-LNP of the present invention and the mRNA-LNP of the model na neocorona vaccine formulation after 6h intramuscular injection into the legs of mice, which are numbered L4-L6.
FIG. 4 shows the results of imaging of mRNA-LNP of the present invention and control formulations including the Modena neo-corona vaccine formulation mRNA-LNP 6h after intramuscular injection into the legs of mice, mouse numbers L7-L9.
FIG. 5 shows the results of imaging of the inventive mRNA-LNP formulation and control formulations including the Modena neo-corona vaccine formulation mRNA-LNP 6h after intramuscular injection into the legs of mice, which are numbered L10-L11.
Detailed Description
The main reagents and suppliers are shown in Table 1
TABLE 1
| Reagent | Suppliers of goods |
| DHA-1 | Sainuobang lattice |
| SM-102 | Sainuobang lattice |
| Plant-derived cholesterol | Sainuobang lattice |
| DMG-PEG2000 | Sainuobang lattice |
| DSPE-PEG2000 | Sainuobang lattice |
| Anhydrous ethanol | Chinese medicine |
| DODAP | Ai Wei Tuo |
| DODMA | Ai Wei Tuo |
| DlinMC3 | Ai Wei Tuo |
| PBS buffer | Shanghai worker |
| Citric acid | Chinese medicine |
| Citric acid sodium salt | Chinese medicine |
| Triton-X100 | Shanghai worker |
| Quant-it™ RiboGreen RNA Assay Kit | Saimei fly |
Example 1
S1, a 5mM pH3.0 citric acid buffer solution containing firefly luciferase (fluc) mRNA was prepared as an aqueous phase, and the mRNA concentration was 36. Mu.g/ml.
S2, preparing absolute ethanol solution of cationic lipid DHA-1, plant-derived cholesterol and phospholipid polyethylene glycol derivative DMG-PEG2000 as mother liquor, wherein the concentrations are 10mg/ml, 10mg/ml and 2mg/ml respectively. According to the DHA-1: plant-derived cholesterol: the molar ratio of DMG-PEG2000 is 20:25:1,60: 70:5,45: 1 preparing a lipid mixed solution as an organic phase.
S3, mixing the two solutions in a microfluidic chip through a microfluidic device, and preparing mRNA lipid nanoparticles by combining positively charged cationic lipids with negatively charged mRNA (the step ofmRNA-LNP) The process is shown in figure 1: the water phase solution and the organic phase solution respectively enter from two sides and a middle flow path of the microfluidic chip, the total flow rate of the water phase is controlled at 3ml/min, the flow rate of the organic phase is controlled at 1ml/min, and the flow rate of the water phase: organic phase flow ratio 3:1, the two-phase flow path is mixed after being converged in the chip, and mRNA and lipid are spontaneously mixedBinding forms mRNA-LNP.
S4, the obtained mRNA-LNP is diluted 10 times with 10mM PBS buffer solution (pH7.0), and purified by ultrafiltration and concentration through an ultrafiltration tube with a 100KD filter pore size. The ultrafiltration mode is 30 degrees fixed angle rotor, centrifugal force 2000g,25 degrees room temperature centrifugal concentration to 1/10 volume.
S5, mRNA-LNP is filtered through a 0.22 mu m filter membrane and stored at 2-8 ℃.
Example 2
S1, a 5mM pH3.0 citric acid buffer solution containing firefly luciferase (fluc) mRNA was prepared as an aqueous phase, and the mRNA concentration was 36. Mu.g/ml.
S2, preparing absolute ethanol solution of cationic lipid DHA-1, plant-derived cholesterol and phospholipid polyethylene glycol derivative DSPE-PEG2000 as mother liquor, wherein the concentrations are 10mg/ml, 10mg/ml and 2mg/ml respectively. According to the DHA-1: plant-derived cholesterol: the molar ratio of DSPE-PEG2000 is 50.38.5, and the nitrogen-phosphorus ratio (N: P) of cationic lipid DHA-1 and mRNA is 5:1 preparing a lipid mixed solution as an organic phase.
S3, mixing the two solutions in a microfluidic chip through a microfluidic device, and preparing mRNA lipid nanoparticles by combining positively charged cationic lipids with negatively charged mRNA (the step ofmRNA-LNP) The process is shown in figure 1: the water phase and the organic phase solution respectively enter from two sides and a middle flow path of the microfluidic chip, the total flow rate of the water phase is controlled at 3ml/min, the flow rate of the organic phase is controlled at 1ml/min, and the water phase: organic phase flow ratio 3:1, the two-phase flow paths are converged and mixed in the chip, and mRNA and lipid are spontaneously combined to form mRNA-LNP.
S4, the obtained mRNA-LNP is diluted 10 times with 10mM PBS buffer solution with pH7.0, and purified by ultrafiltration concentration through an ultrafiltration tube with a 100KD filter pore size. The ultrafiltration mode is 30 degrees fixed angle rotor, centrifugal force 2000g,25 ℃ room temperature centrifugal concentration to 1/10 volume.
S5, mRNA-LNP is filtered through a 0.22 μm filter and stored at 2-8 ℃.
Example 3
S1, a 5mM pH3.0 citric acid buffer solution containing firefly luciferase (fluc) mRNA was prepared as an aqueous phase, and the mRNA concentration was 36. Mu.g/ml.
S2, preparing absolute ethanol solution of cationic lipid DlinMC3, plant-derived cholesterol and phospholipid polyethylene glycol derivative DMG-PEG2000 as mother liquor, wherein the concentrations are 10mg/ml, 10mg/ml and 2mg/ml respectively. According to DlinMC3: plant-derived cholesterol: molar ratio of DMG-PEG2000 50:38.5:1.5, and the nitrogen to phosphorus ratio (N: P) of the cationic lipid DlinMC3 to mRNA 5:1 preparing a lipid mixed solution as an organic phase.
S3, mixing the two solutions in a microfluidic chip through a microfluidic device, and preparing mRNA lipid nanoparticles by combining positively charged cationic lipids with negatively charged mRNA (the step ofmRNA-LNP) The process is shown in figure 1: the water phase and the organic phase solution respectively enter from two sides and a middle flow path of the microfluidic chip, the total flow rate of the water phase is controlled at 3ml/min, the flow rate of the organic phase is controlled at 1ml/min, and the water phase: organic phase flow ratio 3:1, the two-phase flow paths are converged and mixed in the chip, and mRNA and lipid are spontaneously combined to form mRNA-LNP.
S4, the obtained mRNA-LNP is diluted 10 times with 10mM PBS buffer solution with pH7.0, and purified by ultrafiltration concentration through an ultrafiltration tube with a 100KD filter pore size. The ultrafiltration mode is 30 degrees fixed angle rotor, centrifugal force 2000g,25 ℃ room temperature centrifugal concentration to 1/10 volume.
S5, mRNA-LNP is filtered through a 0.22 mu m filter membrane and stored at 2-8 ℃.
Example 4
S1, a 5mM pH3.0 citric acid buffer solution containing firefly luciferase (fluc) mRNA was prepared as an aqueous phase, and the mRNA concentration was 36. Mu.g/ml.
S2, preparing an absolute ethanol solution of cationic lipid DODAP, plant-derived cholesterol and phospholipid polyethylene glycol derivative DMG-PEG2000 as a mother solution, wherein the concentrations are 10mg/ml, 10mg/ml and 2mg/ml respectively. According to DODAP: plant-derived cholesterol: molar ratio of DMG-PEG2000 50:38.5:1.5, and the nitrogen to phosphorus ratio (N: P) of the cationic lipid DODAP to mRNA 5:1 preparing a lipid mixed solution as an organic phase.
S3, mixing the two solutions in a microfluidic chip through a microfluidic device, and combining positively charged cationic lipid with negatively charged mRNA to prepare mRNA lipidNanoparticles of substance (A), (B)mRNA-LNP) The process is shown in figure 1: the water phase and the organic phase solution respectively enter from two sides and a middle flow path of the microfluidic chip, the total flow rate of the water phase is controlled at 3ml/min, the flow rate of the organic phase is controlled at 1ml/min, and the water phase: organic phase flow ratio 3:1, the two-phase flow paths are converged and mixed in the chip, and mRNA and lipid are spontaneously combined to form mRNA-LNP.
S4, the obtained mRNA-LNP is diluted 10 times with 10mM PBS buffer solution (pH7.0), and purified by ultrafiltration and concentration through an ultrafiltration tube with a 100KD filter pore size. The ultrafiltration mode is 30 degrees fixed angle rotor, centrifugal force 2000g,25 degrees room temperature centrifugal concentration to 1/10 volume.
S5, mRNA-LNP is filtered through a 0.22 mu m filter membrane and stored at 2-8 ℃.
Example 5
S1, a 5mM pH3.0 citric acid buffer solution containing firefly luciferase (fluc) mRNA was prepared as an aqueous phase, and the mRNA concentration was 36. Mu.g/ml.
S2, preparing absolute ethanol solution of cationic lipid DODMA, plant-derived cholesterol and phospholipid polyethylene glycol derivative DMG-PEG2000 as mother liquor, wherein the concentrations are 10mg/ml, 10mg/ml and 2mg/ml respectively. According to DODMA: plant-derived cholesterol: molar ratio of DMG-PEG2000 50:38.5:1.5, and the nitrogen to phosphorus ratio (N: P) of cationic lipid DODMA and mRNA 5:1 preparing a lipid mixed solution as an organic phase.
S3, mixing the two solutions in a microfluidic chip through a microfluidic device, and preparing mRNA lipid nanoparticles by combining positively charged cationic lipids with negatively charged mRNA (the step ofmRNA-LNP) The process is shown in figure 1: the water phase and the organic phase solution respectively enter from two sides and a middle flow path of the microfluidic chip, the total flow rate of the water phase is controlled at 3ml/min, the flow rate of the organic phase is controlled at 1ml/min, and the water phase: organic phase flow ratio 3:1, the two-phase flow paths are converged and mixed in the chip, and mRNA and lipid are spontaneously combined to form mRNA-LNP.
S4, the obtained mRNA-LNP is diluted 10 times with 10mM PBS buffer solution with pH7.0, and purified by ultrafiltration concentration through an ultrafiltration tube with a 100KD filter pore size. The ultrafiltration mode is 30 degrees fixed angle rotor, centrifugal force 2000g,25 ℃ room temperature centrifugal concentration to 1/10 volume.
S5, mRNA-LNP is filtered through a 0.22 mu m filter membrane and stored at 2-8 ℃.
Example 6
S1, a 5mM pH3.0 citric acid buffer solution containing firefly luciferase (fluc) mRNA was prepared as an aqueous phase, and the mRNA concentration was 36. Mu.g/ml.
S2, respectively preparing absolute ethyl alcohol solutions of cationic lipid SM-102, plant-derived cholesterol and phospholipid polyethylene glycol derivative DMG-PEG2000 as mother solutions, wherein the concentrations are respectively 10mg/ml, 10mg/ml and 2mg/ml. According to SM-102: plant-derived cholesterol: molar ratio of DMG-PEG2000 50:38.5:1.5, and the nitrogen-to-phosphorus ratio (N: P) of the cationic lipid SM-102 to mRNA 5:1 preparing a lipid mixed solution as an organic phase.
S3, mixing the two solutions in a microfluidic chip through a microfluidic device, and preparing mRNA lipid nanoparticles by combining positively charged cationic lipids with negatively charged mRNA (the step ofmRNA-LNP) The process is shown in figure 1: the water phase and the organic phase solution respectively enter from two sides and a middle flow path of the microfluidic chip, the total flow rate of the water phase is controlled at 3ml/min, the flow rate of the organic phase is controlled at 1ml/min, and the water phase: organic phase flow ratio 3:1, the two-phase flow paths are converged and mixed in the chip, and mRNA and lipid are spontaneously combined to form mRNA-LNP.
S4, the obtained mRNA-LNP is diluted 10 times with 10mM PBS buffer solution with pH7.0, and purified by ultrafiltration concentration through an ultrafiltration tube with a 100KD filter pore size. The ultrafiltration mode is 30 degrees fixed angle rotor, centrifugal force 2000g,25 ℃ room temperature centrifugal concentration to 1/10 volume.
S5, mRNA-LNP is filtered through a 0.22 mu m filter membrane and stored at 2-8 ℃.
Example 7
S1, a 5mM pH3.0 citric acid buffer solution containing firefly luciferase (fluc) mRNA was prepared as an aqueous phase, and the mRNA concentration was 36. Mu.g/ml.
S2, respectively preparing absolute ethyl alcohol solutions of cationic lipid SM-102, DSPC, plant-derived cholesterol and phospholipid polyethylene glycol derivative DMG-PEG2000 as mother solutions, wherein the concentrations are respectively 10mg/ml, 10mg/ml and 2mg/ml. According to SM-102: DSPC: plant-derived cholesterol: molar ratio of DMG-PEG2000 50:10:38.5:1.5, and the nitrogen-to-phosphorus ratio (N: P) of the cationic lipid SM-102 to mRNA 5:1 preparing a lipid mixed solution as an organic phase.
S3, mixing the two solutions in a microfluidic chip through a microfluidic device, and preparing mRNA lipid nanoparticles by combining positively charged cationic lipids with negatively charged mRNA (the step ofmRNA-LNP) The process is shown in figure 1: the water phase and the organic phase solution respectively enter from two sides and a middle flow path of the microfluidic chip, the total flow rate of the water phase is controlled at 3ml/min, the flow rate of the organic phase is controlled at 1ml/min, and the water phase: organic phase flow ratio 3:1, the two-phase flow paths are converged and mixed in the chip, and mRNA and lipid are spontaneously combined to form mRNA-LNP.
S4, the obtained mRNA-LNP is diluted 10 times with 10mM PBS buffer solution with pH7.0, and purified by ultrafiltration concentration through an ultrafiltration tube with a 100KD filter pore size. The ultrafiltration mode is 30 degrees fixed angle rotor, centrifugal force 2000g,25 degrees room temperature centrifugal concentration to 1/10 volume.
S5, mRNA-LNP is filtered through a 0.22 μm filter and stored at 2-8 ℃.
Example 8 mRNA-LNP particle size potential determination
The average particle size and potential of mRNA-LNP samples from examples 1-7 after ultrafiltration through a 100KD filter and filtration through a 0.22 μm filter were determined three times for each sample using a dynamic light scattering nanometer particle size analyzer, as shown in Table 2 below
Table 2 examples 1-7 mRNA-LNP particle size and potential
Example 9 envelope Rate determination of mRNA-LNP
The encapsulation efficiency of mRNA-LNP samples from examples 1-7 after ultrafiltration through a 100KD filter and filtration through a 0.22 μm filter was determined using the Quant-it RiboGreen RNA Assay Kit.
The specific operation is as follows:
(1) The prepared mRNA-LNP suspension and PBS (negative control, equal volume of TE buffer) were diluted to 4 ng/. Mu.l with TE buffer in the kit to obtain mRNA-LNP working solution.
(2) The mRNA-LNP working solution was further diluted with TE buffer (or TE buffer containing 2% Triton-X100) at equal volume, and left to stand at 37 ℃ for 10min after mixing (TE buffer without Triton-X100 was used for determination of unencapsulated free mRNA, while TE buffer containing 2% Triton-X100 was used for determination of Total mRNA in the mRNA-LNP working solution, including free mRNA and mRNA encapsulated in lipid nanoparticles).
(3) After calibration of fluorescence intensity with standards: after the standard curve of the concentration, a proper amount of Quant-it chamber RiboGreen RNA reagent nucleic acid dye is absorbed according to the instruction of the kit and added into each group of samples, the samples are dyed for 5min, each group of dyed samples are moved into an enzyme-labeling instrument for detection, and the mRNA in the samples is accurately quantified by using software.
(4) The mRNA encapsulation efficiency in the lipid nanoparticle was calculated using the following formula.
Encapsulation efficacy = [1-m (free mRNA): m (total mRNA) ]. Times.100% ]
The results are shown in table 3 below:
TABLE 3 examples 1-7 mRNA-LNP encapsulation efficiency
Example 10 verification of the efficacy of the delivery System by in vivo animal experiments
The samples of examples 1-7 were LNP preparations containing the same batch of mRNA expressing firefly luciferase (fluc) prepared by the same microfluidics method, and were injected simultaneously into the leg muscles of Balb/c mice at an injection dose of 4 μ g mRNA/mouse, and the fluorescence intensity at the injection site was measured by an animal imaging device after 6 hours to compare the delivery effects of the different formulations.
The dosage form of the present invention is the mRNA-LNP prepared in example 1, i.e. DHA-1, plant-derived cholesterol, DMG-PEG2000 (molar ratio 20.
Example 2 is a control formulation, using the commonly used commercial phospholipid polyethylene glycol derivative DSPE-PEG2000, using the same 3-component formulation as the formulation of the invention, namely DHA-1, plant-derived cholesterol, DSPE-PEG2000 (molar ratio 50.
Examples 3-5 are control dosage forms using the commonly used commercial cationic lipids dlimc 3, DODAP, DODMA, using the same 3-component formulation as the dosage forms of the present invention, namely dlimc 3, plant-derived cholesterol, DMG-PEG2000 (molar ratio 50; DODAP, plant-derived cholesterol, DMG-PEG2000 molar ratio (50; DODMA, plant-derived cholesterol, DMG-PEG2000 molar ratio (50.
Example 6 is a control formulation, using the main component of the new corona vaccine formulation marketed by Modena, but without the co-phospholipid DSPC, i.e. SM-102, plant-derived cholesterol, DMG-PEG2000 (molar ratio 50.
Example 7 is a control formulation, a new corona vaccine formulation marketed by Modena: SM-102, helper phospholipid (DSPC), plant-derived cholesterol, DMG-PEG2000 (molar ratio 50.
The mRNA-LNP of examples 1 to 7 was photographed by imaging animals 6h after the injection on Balb/c mice, as shown in FIGS. 2 to 5, and the fluorescence intensity photon counts at the injection sites are shown in Table 4.
TABLE 4 example 1-7 fluorescence intensity photon counting of mRNA-LNP on Balb/c mice
From fig. 2 and table 4 it can be found that:
the composition of the ionizable cationic lipid (DHA-1), the plant-derived cholesterol and the phospholipid polyethylene glycol derivative (DMG-PEG 2000) of the dosage form of the invention is characterized in that the molar ratio of (20-60): (25-70): (1-5), preferably (45-55): (35-40): (1-5), all of which have excellent in vivo mRNA delivery effects.
In comparison with the control formulation in example 2, the commonly-used commercial phospholipid polyethylene glycol derivative DSPE-PEG2000 is used to replace the DMG-PEG2000 used in the invention, and the in vivo transfection effect is remarkably reduced under the condition that the formulation of the other components is not changed, which shows that the effect of the invention is better than that of the traditional formulation by using DMG-PEG 2000.
The control formulation of comparative example 3, using the commonly used commercially available DlinMC3 as the ionizable lipid, showed slightly lower in vivo transfection efficiency than the formulation of the present invention without formulation changes of the remaining components.
In the control formulation of comparative example 4, with the usual commercially available DODAP as ionizable lipid, the in vivo transfection efficiency was significantly lower than in the formulation of the present invention with the remaining components being formulated unchanged.
The control formulation of comparative example 5, using the commonly used commercially available DODMA as the ionizable lipid, showed significantly less in vivo transfection efficiency than the formulation of the present invention without formulation changes for the remaining components.
The control formulation of comparative example 6, which was prepared from the ionizable lipid SM-102 of the new crown vaccine formulation marketed by Modena corporation, had an in vivo transfection effect comparable to that of the formulation of the present invention, with the remaining components formulated unchanged (3-component formulation, without the auxiliary phospholipid).
Compared with the control dosage form of example 7, a new crown vaccine dosage form marketed by the company Modena is adopted: SM-102, helper phospholipid (DSPC), plant-derived cholesterol, DMG-PEG2000 (50 molar ratio 50. Meanwhile, compared with the control formulation in example 6, it can be seen that the transfection effect can be remarkably improved by removing the auxiliary phospholipid in the 3-component preparation compared with the 4-component preparation. This is probably due to the development of early mRNA-LNP formulation formulations, which require co-phospholipids to enhance endosome escape capacity due to the use of ionizable cationic lipids typified by permanently ionized cationic lipids (e.g., DOTAP) or preliminary DODAP, and the unique compound structure design of the latest ionizable cationic lipids, now typified by SM-102 and DHA-1 used in the present invention, provides delivery effects far superior to previous cationic lipid compounds, and thus the effects of any improved optimized co-phospholipids are not relatively deteriorated; the components of the helper phospholipid may interfere with the delivery effect of the ionizable lipid. Therefore, the auxiliary phospholipid is removed, and the in vivo transfection effect is greatly improved.
Claims (14)
1. An mRNA drug delivery system, which is characterized in that the mRNA drug delivery system is a lipid nanoparticle loaded with one or more mRNAs; the lipid nanoparticles are prepared from ionizable cationic lipid DHA-1, cholesterol and phospholipid polyethylene glycol derivative DMG-PEG 2000; wherein, the molar ratio of the ionizable cationic lipid DHA-1 to the cholesterol to the phospholipid polyethylene glycol derivative DMG-PEG2000 is 25-60:25-70: 1-5.
2. The delivery system according to claim 1, characterized in that the cholesterol is selected from plant-derived cholesterol.
3. The delivery system according to claim 1, characterized in that the lipid nanoparticle has an average particle size of 60-140nm.
4. The delivery system of claim 1, wherein the mRNA drug delivery system is prepared by microfluidic devices.
5. The delivery system according to claim 1, characterized in that under neutral environmental conditions the Zeta potential of the mRNA-lipid nanoparticle is from-15 mV to +5mV.
6. A method of preparing the mRNA drug delivery system of claim 1, characterized by comprising the steps of:
s1, preparing an mRNA solution as a water phase;
s2, preparing an absolute ethanol solution containing all components of the lipid nanoparticles as an organic phase, wherein the molar ratio of ionizable cationic lipid DHA-1 to cholesterol to phospholipid polyethylene glycol derivative DMG-PEG2000 is 25-60:25-70: 1-5, the nitrogen-phosphorus ratio of the ionizable cationic lipid DHA-1 to mRNA is 3-8:1;
and S3, controlling the flow rate ratio and the flow velocity of the aqueous phase and the organic phase by adopting a microfluidic method, mixing the two solutions in a microfluidic chip, and combining positively charged cationic lipid with negatively charged mRNA to prepare the mRNA lipid nanoparticles.
7. The method of claim 6, wherein the mRNA solution in S1 is prepared by dissolving mRNA in a buffer selected from the group consisting of 5-10mM citrate buffer at pH 3.0.
8. The method according to claim 6, wherein in S2, the ratio of nitrogen to phosphorus of the cationic lipid DHA-1 to mRNA is 5:1 preparing a lipid mixed solution as an organic phase.
9. The method according to claim 6, wherein in S3, the flow rate of the aqueous phase is 3ml/min and the flow rate of the organic phase is 1ml/min.
10. The method of claim 6, further comprising purifying the S4, mRNA-lipid nanoparticles by ultrafiltration using an ultrafiltration tube; the size of the retention pore is 100kd.
11. Use of the delivery system of claim 1 in the preparation of a mRNA medicament.
12. The use according to claim 11, wherein the mRNA is a complete mRNA molecule of a functional protein, a therapeutic monoclonal antibody, a B cell epitope, a T cell epitope or a tumor neoantigen peptide fragment expressed for the treatment of a disease.
13. Use of the delivery system of claim 1 in the preparation of an mRNA vaccine.
14. The use according to claim 13, wherein the mRNA is a complete mRNA molecule of a functional protein, a therapeutic monoclonal antibody, a B cell epitope, a T cell epitope or a tumor neoantigen peptide fragment expressed in a prophylactic manner.
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