Cell membrane encapsulated lipid nanoparticle, preparation method therefor, and use thereof

Abstract

The present invention relates to a cell membrane encapsulated lipid nanoparticle, a preparation method therefor and the use thereof. The cell membrane encapsulated lipid nanoparticle comprises a lipid nanoparticle and a platelet cell membrane, the surface of the lipid nanoparticle being coated with the platelet cell membrane. Coating the surface of the lipid nanoparticle with the platelet cell membrane can enable a drug to be effectively targeted to and retain in a focus area, thereby improving the targeting property; and the biocompatibility of the platelet cell membrane can also protect the lipid nanoparticle from being rapidly cleared by the body, thereby improving the delivery efficiency. The present invention has a good application prospect in nucleic acid drug delivery.

Description

Cell membrane encapsulated lipid nanoparticles and preparation method and application thereof TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a cell membrane encapsulated lipid nanoparticle and a preparation method and application thereof. BACKGROUND

[0002] The great success of messenger ribonucleic acid (mRNA) technology in vaccine research and development directly accelerates the development of vaccines and solves some technical bottlenecks of the application of mRNA technology in vaccine research and development. The application of nucleic acid drugs (mRNA, siRNA, ASO, etc.) in the development of new types of therapeutic and preventive tumor vaccines brings new opportunities for tumor treatment.

[0003] Nucleic acid drugs have poor stability in vivo and are easily cleared, and therefore, a drug delivery system is often used to maintain the structure and activity of nucleic acids, and also effectively target lesion areas. The mainstream technology at present is to use lipid nanoparticles (LNP) to encapsulate nucleic acids and perform drug delivery.

[0004] In the prior art, the technology of delivering nucleic acid drugs (such as messenger ribonucleic acid (mRNA), small interfering ribonucleic acid (siRNA) and antisense nucleotides (ASO), etc.) based on lipid nanoparticles (LNP) has the following deficiencies: 1) the polyethylene glycolated lipid (PEG lipid) in the formula can activate anti-PEG antibodies, cause hypersensitivity, accelerate blood clearance, and cause systemic immunogenicity after repeated administration, resulting in rapid clearance of the drug and failure to produce sufficient efficacy; 2) LNP can adsorb serum proteins and accumulate in the liver, causing local toxicity, and has poor tumor tissue targeting, and after intratumoral injection, it can still spread to adjacent tissues and organs.

[0005] SUMMARY

[0006] Based on the above technical background, the main purpose of the present application is to provide a cell membrane encapsulated lipid nanoparticle and a preparation method and application thereof to overcome the deficiencies in the prior art.

[0007] To achieve the aforementioned purposes, the technical solutions adopted by the present application include:

[0008] The first aspect of the present application is to provide a cell membrane encapsulated lipid nanoparticle, which comprises a lipid nanoparticle and a platelet cell membrane, and the platelet cell membrane is coated on the surface of the lipid nanoparticle.

[0009] The mass ratio of the lipid nanoparticle to the platelet cell membrane is (5-20):1.

[0010] Preferably, the mass ratio of the lipid nanoparticle to the platelet cell membrane is 5:1.

[0011] Preferably, the lipid nanoparticles are prepared from a lipid mixture and an mRNA solution, and the volume ratio of the lipid mixture to the mRNA solution is 1:(2-6).

[0012] More preferably, the lipid mixture is prepared from ionizable lipid, cationic lipid, distearoylphosphatidylcholine (DSPC), cholesterol and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethyleneglycol (DMG-PEG2000) at a molar ratio of 15-45:15-45:5-15:20-30:0.5-1.5.

[0013] wherein:

[0014] The ionizable lipid is ((4-hydroxybutyl)azabicyclohexyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), 4-(N,N-dimethylamino)butanoic acid, dilinoleylmethyl ester (DLin-MC3-DMA) or 1-octylnonyl 8-[(2-hydroxyethyl)[6-O-6-(undecyloxy)hexyl]amino]-octanoate (SM-102).

[0015] The cationic lipid is (2,3-dioleyloxypropyl)trimethylammonium chloride (DOTAP) or DC-Cholesterol.

[0016] The second aspect of the present application provides a method for preparing the cell membrane encapsulated lipid nanoparticles of the first aspect of the present application, the method comprising:

[0017] The lipid nanoparticles and the platelet cell membrane are mixed, and the mixed solution is first extruded through a filter membrane with a pore size of 0.2-1 μm, and then extruded through a filter membrane with a pore size of 0.1-0.4 μm, to obtain the cell membrane encapsulated lipid nanoparticles.

[0018] Preferably, the method for preparing the lipid nanoparticles comprises:

[0019] Step 1, dissolving the lipids in anhydrous ethanol to obtain a lipid mixture;

[0020] Step 2, dissolving the mRNA in a citric acid solution and stirring uniformly to obtain an mRNA solution;

[0021] Step 3, mixing the lipid mixture and the mRNA solution by a microfluidic method to prepare the lipid nanoparticles.

[0022] More preferably, in step 2, the concentration of the citric acid solution is 5-25 mM.

[0023] The concentration of the mRNA solution is 50-500 μg / mL.

[0024] In step 3, the flow rate of the lipid mixture solution is set to 3-7 mL / min.

[0025] The flow rate of the mRNA solution is set to 9-21 mL / min.

[0026] Preferably, the preparation method of the platelet cell membrane comprises:

[0027] The platelet solution is diluted with the HEP buffer, and after centrifugation, the supernatant is discarded, and then the precipitate is resuspended with the lysis buffer, followed by freezing, thawing, centrifugation, discarding the supernatant, and then resuspending the precipitate with the lysis buffer, freezing, thawing, centrifugation, discarding the supernatant, and finally resuspending the precipitate with the lysis buffer, thereby obtaining the platelet cell membrane.

[0028] More preferably, the HEP buffer and the platelet solution are diluted at a mass ratio of (2-4):1.

[0029] After dilution, centrifugation is performed at 1500-2500 rpm for 10-20 min, followed by centrifugation at 3000-5000 rpm for 5-15 min, and then the precipitate is resuspended with the lysis buffer after discarding the supernatant, followed by freezing at -75 to -85°C for 40-50 min, and then thawing at 30-40°C, and after thawing, centrifugation is performed at 25000-35000 rpm for 10-20 min, and then the precipitate is resuspended with the lysis buffer after discarding the supernatant, and then frozen at -75 to -85°C for 40-50 min, and then thawed at 30-40°C, and finally centrifuged at 30000-40000 rpm for 15-25 min.

[0030] The third aspect of the present application provides a use of the cell membrane encapsulated lipid nanoparticle of the first aspect of the present application in drug delivery, particularly for the delivery of nucleic acid drugs.

[0031] The delivered nucleic acid molecules include, but are not limited to, at least one protein, polypeptide, polypeptide fragment encoding a tumor-associated antigen or tumor-specific antigen, a protein, polypeptide, polypeptide fragment expressed to stimulate the body to produce an anti-tumor immune response, or an siRNA, miRNA or ASO that can regulate the expression level of a local cancer or tumor-specific protein, polypeptide, polypeptide fragment.

[0032] The present application has the following beneficial effects:

[0033] (1) The present application can effectively target tumor cells and stay in the lesion area under the action of platelet cell membrane surface proteins and receptors, and the drug will not spread to adjacent tissues and organs; at the same time, the biocompatibility of the platelet cell membrane can also protect the lipid nanoparticles from being rapidly cleared by the body, thereby enhancing the targeting and delivery efficiency of tumor tissues.

[0034] (2) The present application can effectively target tumor cells and stay in the lesion area under the action of platelet cell membrane surface proteins and receptors, and the drug will not spread to adjacent tissues and organs; at the same time, the biocompatibility of the platelet cell membrane can also protect the lipid nanoparticles from being rapidly cleared by the body, thereby enhancing the targeting and delivery efficiency of tumor tissues.

[0035] (3) The cell membrane encapsulated lipid nanoparticle has better biocompatibility, higher delivery efficiency and higher targeting than the lipid nanoparticle, and has good application prospect in nucleic acid drug delivery. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 shows the signal intensity of Examples 1-4 and Comparative Examples 1-2 in three cell lines. DETAILED DESCRIPTION

[0037] The present application will be described in detail below, and the features and advantages of the present application will become clearer and more explicit with these descriptions.

[0038] The existing LNP (lipid nanoparticle) research is mainly in the development of new cationic lipids / ionizable lipids or the adjustment of LNP formula ratio. The innovation of the present application is to encapsulate a cell membrane on the basis of LNP formula, so as to have the effect of targeting tumor cells, and not easily trigger immunogenicity of the body, thereby improving the targeting and delivery efficiency.

[0039] The first aspect of the present application is to provide a cell membrane encapsulated lipid nanoparticle, which comprises a lipid nanoparticle and a platelet cell membrane, and the platelet cell membrane is coated on the surface of the lipid nanoparticle.

[0040] The mass ratio of the lipid nanoparticle to the platelet cell membrane is (5-20): 1, and the mass of the platelet cell membrane is calculated based on the total mass of the protein in the platelet cell membrane.

[0041] Preferably, the mass ratio of the lipid nanoparticle to the platelet cell membrane is 5:1, and the mass of the platelet cell membrane is calculated based on the total mass of the protein in the platelet cell membrane.

[0042] The lipid nanoparticle is prepared from a lipid mixture and an mRNA solution, wherein the volume ratio of the lipid mixture to the mRNA solution is 1:(2-4), preferably the volume ratio is 1:3.

[0043] The lipid mixture is prepared from ionizable lipid, cationic lipid, distearoylphosphatidylcholine (DSPC), cholesterol and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethyleneglycol (DMG-PEG2000) in a molar ratio of 15-45:15-45:5-15:20-30:0.5-1.5.

[0044] Preferably, the lipid mixture is prepared from ionizable lipid, cationic lipid, distearoylphosphatidylcholine (DSPC), cholesterol and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethyleneglycol (DMG-PEG2000) in a molar ratio of 20-30:35-45:7:27:1.1.

[0045] wherein:

[0046] The ionizable lipid is selected from (4-hydroxybutyl)azabicycloalkyl bis(hexan-6,1-diyl) bis(2-hexyldecanoate) (ALC-0315), 4-(N,N-dimethylamino)butanoic acid (dilinoleyl) methyl ester (DLin-MC3-DMA) or 1-octylnonyl 8-[(2-hydroxyethyl)[6-O-6-(undecyloxy)hexyl]amino]-octanoate (SM-102), preferably 1-octylnonyl 8-[(2-hydroxyethyl)[6-O-6-(undecyloxy)hexyl]amino]-octanoate (SM-102).

[0047] The cationic lipid is (2,3-dioleyloxypropyl)trimethylammonium chloride (DOTAP) or DC-Cholesterol, preferably (2,3-dioleyloxypropyl)trimethylammonium chloride (DOTAP).

[0048] The mRNA solution is prepared by dissolving mRNA in a citric acid solution.

[0049] The concentration of the citric acid solution is 5-15 mM, preferably the concentration of the citric acid solution is 10 mM.

[0050] The concentration of the mRNA solution is 50-500 μg / mL, preferably the concentration of the mRNA is 200-300 μg / mL.

[0051] Compared with the prior art lipid nanoparticle, the present application has better biocompatibility, higher delivery efficiency and higher targeting to tumor cells by coating the platelet cell membrane on the surface of the lipid nanoparticle.

[0052] The second aspect of the present application provides a method for preparing the cell membrane encapsulated lipid nanoparticle of the first aspect of the present application, comprising the following steps:

[0053] Step 1, dissolving lipids in anhydrous ethanol to obtain a lipid mixture;

[0054] Step 2, dissolving mRNA in a citric acid solution and stirring uniformly to obtain an mRNA solution;

[0055] Step 3, mixing the lipid mixture and the mRNA solution by microfluidic method to prepare lipid nanoparticles;

[0056] Step 4, diluting the platelet solution with HEP buffer, centrifuging and discarding the supernatant, then resuspending the precipitate with lysis buffer, followed by freezing, thawing, centrifugation, discarding the supernatant, then resuspending the precipitate with lysis buffer, freezing, thawing, centrifugation, discarding the supernatant, and finally resuspending the precipitate with lysis buffer to obtain platelet cell membranes;

[0057] Step 5, mixing the lipid nanoparticles prepared in step 3 and the platelet cell membranes prepared in step 4, and then extruding to obtain cell membrane encapsulated lipid nanoparticles.

[0058] The above steps are described in detail as follows.

[0059] In step 1, the lipids SM102, DOTAP, DSPC, cholesterol (CHO) and DMG-PEG2000 are dissolved in anhydrous ethanol according to a molar ratio of 15-35:30-50:5-10:25-30:0.5-1.5 to obtain a lipid mixture with a total lipid concentration of 5-40 mg / mL.

[0060] Preferably, the lipids SM102, DOTAP, DSPC, cholesterol (CHO) and DMG-PEG2000 are dissolved in anhydrous ethanol according to a molar ratio of 20-30:35-45:7:27:1.1 to obtain a lipid mixture with a total lipid concentration of 5 mg / mL.

[0061] In step 2, the concentration of the citric acid solution is 5-25 mM, and preferably the concentration of the citric acid solution is 10 mM.

[0062] The concentration of the mRNA solution is 50-500 μg / mL, and preferably the concentration of the mRNA is 200-300 μg / mL.

[0063] In step 3, the volume ratio of the lipid mixture to the mRNA solution is 1:(2-6), and preferably the volume ratio is 1:3.

[0064] The mixing is performed by using a microfluidic device. The flow rate of the lipid mixture is set to 3-7 mL / min. Preferably, the flow rate of the lipid mixture is 5 mL / min.

[0065] The flow rate of the mRNA solution is set to 9-21 mL / min. Preferably, the flow rate of the mRNA solution is 15 mL / min.

[0066] The lipid mixture mixed in a specific ratio is dissolved in ethanol, and then mixed with the nucleic acid dissolved in the aqueous phase buffer system by using a microfluidic device or the like to form a nano-sized lipid particle. The cationic lipid / ionizable lipid (DOTAP, SM-102) will adsorb the nucleic acid in the particle based on the charge adsorption effect.

[0067] In step 4, the HEP buffer and the platelet solution are diluted at a mass ratio of (2-4):1. Preferably, the HEP buffer and the platelet solution are diluted at a mass ratio of 3:1.

[0068] After dilution, centrifugation is performed at 1500-2500 rpm for 10-20 min, followed by centrifugation at 3000-5000 rpm for 5-15 min. After discarding the supernatant, the precipitate is resuspended with lysis buffer, followed by freezing at -75 to -85℃ for 40-50 min, then thawing at 30-40℃. After thawing, centrifugation is performed at 25000-35000 rpm for 10-20 min. After discarding the supernatant, the precipitate is resuspended with lysis buffer, then frozen at -75 to -85℃ for 40-50 min, thawed at 30-40℃, and finally centrifuged at 30000-40000 rpm for 15-25 min.

[0069] Preferably, after dilution, centrifugation is performed at 2000 rpm for 15 min, followed by centrifugation at 4000 rpm for 10 min. After discarding the supernatant, the precipitate is resuspended with lysis buffer, then frozen at -80℃ for 45 min, thawed at 37℃, centrifuged at 30000 rpm for 15 min after thawing, resuspended with lysis buffer, then frozen at -80℃ for 45 min, thawed at 37℃, and finally centrifuged at 35000 rpm for 20 min.

[0070] The platelet cell membrane obtained after lysis and purification not only retains its biological activity, but also removes various intracellular substances that may cause immunogenicity in the body. When the platelet cell membrane is used as a protective membrane to coat the surface of the lipid nanoparticle, it can protect the lipid nanoparticle from being rapidly cleared by the body. At the same time, under the action of the proteins and receptors on the surface of the platelet cell membrane, the drug can be effectively targeted to tumor cells and stay in the lesion area, thereby improving the targeting and delivery efficiency.

[0071] In step 5, the mixing ratio of the lipid nanoparticles prepared in step 3 and the platelet cell membrane prepared in step 4 (calculated as protein) is (5-20):1.

[0072] Preferably, the mixing ratio of the lipid nanoparticles prepared in step 3 and the platelet cell membrane prepared in step 4 (calculated as protein) is =5:1.

[0073] After mixing, the solution is first extruded through a filter membrane with a pore size of 0.2-1 μm, and then through a filter membrane with a pore size of 0.1-0.4 μm, preferably first through a filter membrane with a pore size of 0.4-0.8 μm, and then through a filter membrane with a pore size of 0.1-0.2 μm.

[0074] The LNP is mixed with the purified platelet cell membrane at a certain ratio, and the platelet cell membrane is wrapped onto the outer surface of the LNP by microfluidic, membrane extrusion or ultrasonic treatment to form a core-shell structure.

[0075] The third aspect of the present application provides a use of the cell membrane encapsulated lipid nanoparticle of the first aspect of the present application in drug delivery, particularly for the delivery of nucleic acid drugs.

[0076] The delivered nucleic acid molecules include, but are not limited to, at least a protein, polypeptide or polypeptide fragment encoding a tumor-associated antigen or tumor-specific antigen, a protein, polypeptide or polypeptide fragment expressed to stimulate the body to produce an anti-tumor immune response, or an siRNA, miRNA or ASO that can regulate the expression level of a protein, polypeptide or polypeptide fragment with local cancer or tumor characteristics.

[0077] Examples

[0078] The present application is further illustrated by the following specific examples, which are only intended to illustrate the present application and not to limit the scope of the present application. The raw materials used in the examples of the present application are all purchased except for the HEP buffer and the lysis buffer.

[0079] The formula of the HEP buffer is: NaCl 8.182 g / L, KCl 0.201 g / L, HEPES (4-hydroxyethylpiperazine ethanesulfonic acid) 0.907 g / L, EGTA (ethylene glycol bis(2-aminoethyl ether)) 1.902 g / L, prostaglandin E1 1 mg / L.

[0080] The formula of the lysis buffer is: NaCl 3.915 g / L, KCl 0.108 g / L, NaHCO3 0.504 g / L, Na2HPO4 46 mg / mL, MgCl2 48 mg / L, HEPES (4-hydroxyethylpiperazine ethanesulfonic acid) 4.766 g / L, prostaglandin E1 1 mg / L, EDTA 0.372 g / L, NP-40 (ethyl phenyl polyethylene glycol) 106 mg / L. Among them, NP-40 can be replaced with Tween 20, and the concentration of Tween 20 is 555 mg / L.

[0081] Example 1

[0082] Step 1, preparation of lipid mixture: dissolve the lipids SM102, DOTAP, DSPC, cholesterol (CHO) and DMG-PEG2000 in anhydrous ethanol according to the molar ratio of 20:45:7:27:1.1 to prepare a mother liquor with a concentration of 10 mg / mL, the total lipid concentration is 5 mg / mL, and 0.75 mL is prepared.

[0083] Step 2: mRNA solution preparation: dissolve 404 μg of mRNA encoding firefly luciferase (Luciferase) in 4.5 mL of 10 mM citric acid solution (pH 4.0) and stir uniformly.

[0084] Step 3: Preparation of lipid nanoparticles: take 0.75 mL of lipid mixture and 2.25 mL of mRNA solution, mix to prepare lipid nanoparticles using a microfluidic device, set the flow rate of the lipid mixture to 5 mL / min, and the flow rate of the mRNA solution to 15 mL / min.

[0085] Step 4: Purification of cell membrane: dilute the platelet solution with HEP buffer at a ratio of 1:3, centrifuge at 2000 rpm for 15 min, then centrifuge at 4000 rpm for 10 min, discard the supernatant, resuspend the precipitate with lysis buffer, transfer to a -80°C refrigerator, freeze for 45 min, then melt in a 37°C water bath; centrifuge the melted sample at 30000 rpm for 15 min, discard the supernatant, resuspend the precipitate with lysis buffer, transfer to a -80°C refrigerator, freeze for 45 min, then melt in a 37°C water bath; centrifuge the melted sample at 35000 rpm for 20 min, discard the supernatant, resuspend with lysis buffer again, and obtain the purified platelet cell membrane.

[0086] Step 5: Cell membrane encapsulated lipid nanoparticle preparation: The lipid nanoparticle prepared in step 3 and the platelet cell membrane prepared in step 4 were mixed, the mixing ratio was lipid concentration: protein concentration = 5: 1. The mixed material was extruded using an extruder, first extruded from a filter membrane with a pore size of 0.8 μm, and then extruded from a filter membrane with a pore size of 0.2 μm; thus the cell membrane encapsulated lipid nanoparticle PLNP1 was obtained.

[0087] Example 2

[0088] The preparation of the cell membrane encapsulated lipid nanoparticle was carried out in a similar manner as in Example 1, except that in step 5, the mixed material was first extruded from a filter membrane with a pore size of 0.4 μm, and then extruded from a filter membrane with a pore size of 0.1 μm. After the material was extruded, the cell membrane encapsulated lipid nanoparticle PLNP2 was obtained.

[0089] Example 3

[0090] The preparation of the cell membrane encapsulated lipid nanoparticle was carried out in a similar manner as in Example 1, except that in step 1, the lipid mixture was prepared: the lipids SM102, DOTAP, DSPC, cholesterol (CHO) and DMG-PEG2000 were dissolved in anhydrous ethanol according to the molar ratio of 30:35:7:27:1.1, to prepare a mother liquor with a concentration of 10 mg / mL, the total lipid concentration was 5 mg / mL, and 0.75 mL was prepared.

[0091] The cell membrane encapsulated lipid nanoparticle PLNP3 was obtained.

[0092] Example 4

[0093] The preparation of the cell membrane encapsulated lipid nanoparticle was carried out in a similar manner as in Example 1, except that in step 1, the lipid mixture was prepared: the lipids SM102, DOTAP, DSPC, cholesterol (CHO) and DMG-PEG2000 were dissolved in anhydrous ethanol according to the molar ratio of 30:35:7:27:1.1, to prepare a mother liquor with a concentration of 10 mg / mL, the total lipid concentration was 5 mg / mL, and 0.75 mL was prepared.

[0094] In step 5, the mixed material was first extruded from a filter membrane with a pore size of 0.4 μm, and then extruded from a filter membrane with a pore size of 0.1 μm. After the material was extruded, the cell membrane encapsulated lipid nanoparticle PLNP2 was obtained.

[0095] Comparative Example

[0096] Comparative Example 1

[0097] Step 1, Lipid solution preparation: Dissolve the lipids SM102, DOTAP, DSPC, Cholesterol (CHO) and DMG-PEG2000 in anhydrous ethanol according to the molar percentage SM102:DOTAP:DSPC:CHO:DMG-PEG2000 of 20:45:7:27:1.1, prepare a stock solution with a concentration of 10 mg / mL, the total lipid concentration is 5 mg / mL, prepare 0.75 mL.

[0098] Step 2, mRNA solution preparation: Dissolve 404 μg of mRNA encoding firefly luciferase (Luciferase) in 4.5 mL of 10 mM citric acid solution (pH 4.0) and stir evenly.

[0099] Step 3, Preparation of lipid nanoparticles: Take 0.75 mL of lipid solution and 2.25 mL of mRNA solution, mix using a microfluidic device to prepare lipid nanoparticles LNP1, set the flow rate of the lipid solution to 5 mL / min, and the flow rate of the mRNA solution to 15 mL / min.

[0100] Comparative Example 2

[0101] Step 1, Lipid solution preparation: Dissolve the lipids SM102, DOTAP, DSPC, Cholesterol (CHO) and DMG-PEG2000 in anhydrous ethanol according to the molar percentage SM102:DOTAP:DSPC:CHO:DMG-PEG2000 of 20:45:7:27:1.1, prepare a stock solution with a concentration of 10 mg / mL, the total lipid concentration is 5 mg / mL, prepare 0.75 mL.

[0102] Step 2, mRNA solution preparation: Dissolve 404 μg of mRNA encoding firefly luciferase (Luciferase) in 4.5 mL of 10 mM citric acid solution (pH 4.0) and stir evenly.

[0103] Step 3, Preparation of lipid nanoparticles: Take 0.75 mL of lipid solution and 2.25 mL of mRNA solution, mix using a microfluidic device to prepare lipid nanoparticles LNP2, set the flow rate of the lipid solution to 5 mL / min, and the flow rate of the mRNA solution to 15 mL / min.

[0104] Experimental Example

[0105] Main index test of experimental example 1

[0106] The cell membrane encapsulated lipid nanoparticles prepared in Examples 1-4 and the lipid nanoparticles prepared in Comparative Examples 1-2 were respectively subjected to particle size, polydispersity index and zeta potential, mRNA concentration and encapsulation efficiency tests. The particle size, polydispersity index and zeta potential were tested by dynamic light scattering method, and the equipment was ZetaSizer Pro from Malvern. The mRNA concentration and encapsulation efficiency were tested by Ribogreen detection kit from Thermo. The test results are shown in Table 1.

[0107] Table 1 sample test results

[0108] As can be seen from Table 1, the particle size of the cell membrane encapsulated lipid nanoparticles is larger than that of the unencapsulated lipid nanoparticles. The particle size of the cell membrane encapsulated lipid nanoparticles is 189.1-211.6 nm. The polydispersity index and zeta potential of the cell membrane encapsulated lipid nanoparticles are greatly improved, and the mRNA concentration is reduced.

[0109] Experimental Example 2 in vitro transfection experiment

[0110] The cell membrane encapsulated lipid nanoparticles prepared in Examples 1-4 and the lipid nanoparticles prepared in Comparative Examples 1-2 were respectively subjected to in vitro transfection experiments.

[0111] The specific operation process is as follows: first, three cell lines (HEK-293T, Hela, HepG2) were respectively digested with 0.25% trypsin and counted. The cell concentration was adjusted to 3x10 5 cells / mL, and 100 μL of cell solution was added to each well of a 96-well cell culture plate, so that the number was 3x10 4 The cell culture plate was placed in an incubator and cultured at 37°C, 5% CO2 overnight (16-18h). Then the cell membrane encapsulated lipid nanoparticles prepared in Examples 1-4 and the lipid nanoparticles prepared in Comparative Examples 1-2 were diluted with DMEM medium to a mRNA concentration of 200 ng / mL, and the diluted samples were added to the corresponding cell well plates at 100 μL / well, i.e. the actual added mRNA was 20 ng. The plates were placed in an incubator and cultured at 37°C, 5% CO2 for 24h. The cell culture plate was taken out and equilibrated to room temperature (25°C). Then 100 μL of luciferase detection reagent was added to each well, shaken for 30s and then placed for 3min. The plate was read in an enzyme marker. The results are shown in Table 2 and Figure 1.

[0112] Table 2

[0113] The results show that, compared with Comparative Example 1 (LNP1), after being coated by two different extrusion processes, Example 1 (PLNP1) and Example 2 (PLNP2), the signal intensity of luciferase in the three cell lines after in vitro transfection is significantly improved, with an average increase of 4.4 times and 2.6 times, respectively, under the same mRNA addition amount; for Comparative Example 2 (LNP2), after being coated by two different processes, Example 3 (PLNP3) and Example 4 (PLNP4), the signal intensity of luciferase in the three cell lines after in vitro transfection is significantly improved, with an average increase of 6.1 times and 5.2 times, respectively, under the same mRNA addition amount. It is shown that the efficiency of in vitro transfection is significantly improved after the cell membrane is coated with lipid nanoparticles.

[0114] The above detailed description of the application is made in conjunction with the specific embodiments and exemplary examples, but these descriptions cannot be understood as limiting the application. Those skilled in the art understand that various equivalent substitutions, modifications or improvements can be made to the technical solutions and embodiments of the application without departing from the spirit and scope of the application, and these all fall within the scope of the application. The scope of protection of the application is subject to the appended claims.

Claims

1. A cell membrane-encapsulated lipid nanoparticle, characterized in that, The cell membrane-encapsulated lipid nanoparticles include lipid nanoparticles and platelet cell membranes, with the platelet cell membranes coating the surface of the lipid nanoparticles. The mass ratio of lipid nanoparticles to platelet cell membranes is (5-20):1, and the mass of platelet cell membranes is calculated as the total mass of proteins in the platelet cell membrane.

2. The cell membrane-encapsulated lipid nanoparticles according to claim 1, characterized in that, The mass ratio of lipid nanoparticles to platelet cell membranes was 5:1, and the mass of platelet cell membranes was calculated as the total mass of proteins in the platelet cell membranes.

3. The cell membrane-encapsulated lipid nanoparticles according to claim 1, characterized in that, The lipid nanoparticles were prepared by a lipid mixture and an mRNA solution, with a volume ratio of 1:(2-6).

4. The cell membrane-encapsulated lipid nanoparticles according to claim 3, characterized in that, The lipid mixture is prepared from ionizable lipids, cationic lipids, distearate phosphatidylcholine, cholesterol, and 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol in a molar ratio of 15–45:15–45:5–15:20–30:0.5–1.

5. The ionizable lipids are ((4-hydroxybutyl)azadialkyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), 4-(N,N-dimethylamino)butyrate (dilinoleyl) methyl ester, or 1-octylnonyl 8-[(2-hydroxyethyl)[6-O-6-(undecyloxy)hexyl]amino]octanoate; The cationic lipids are (2,3-dioleoxypropyl)trimethylammonium chloride or DC-cholesterol.

5. A method for preparing cell membrane-encapsulated lipid nanoparticles according to any one of claims 1 to 4, characterized in that, The method includes: Lipid nanoparticles and platelet cell membranes were mixed, and the mixture was first... Cell membrane-encapsulated lipid nanoparticles are obtained by extruding from a filter membrane with a pore size of 0.2–1 μm and then from a filter membrane with a pore size of 0.1–0.4 μm.

6. The method according to claim 5, characterized in that, The method for preparing the lipid nanoparticles includes: Step 1: Dissolve the lipids in anhydrous ethanol to obtain a lipid mixture; Step 2: Dissolve the mRNA in citric acid solution and stir until homogeneous to obtain an mRNA solution; Step 3: The lipid mixture and mRNA solution are mixed using a microfluidic method to obtain lipid nanoparticles.

7. The method according to claim 6, characterized in that, In step 2, the concentration of the citric acid solution is 5–25 mM, and the concentration of the mRNA solution is 50–500 μg / mL; In step 3, the flow rate of the lipid mixture is set to 3–7 mL / min; Set the flow rate of the mRNA solution to 9–21 mL / min.

8. The method according to claim 5, characterized in that, The method for preparing the platelet cell membrane includes: The platelet solution was diluted with HEP buffer, centrifuged and the supernatant was discarded. The precipitate was then resuspended with lysis buffer, followed by freezing, thawing, centrifugation, and discarding of the supernatant. The precipitate was then resuspended with lysis buffer again. After freezing, thawing, centrifugation, and discarding of the supernatant, the precipitate was finally resuspended with lysis buffer to obtain the platelet cell membrane.

9. The method according to claim 8, characterized in that, The HEP buffer and platelet solution were diluted at a volume ratio of (2-4):1; After dilution, centrifuge at 1500–2500 rpm for 10–20 min, followed by centrifugation at 3000–5000 rpm for 5–15 min. Discard the supernatant, resuspend the precipitate in lysis buffer, then freeze at -75–-85°C for 40–50 min, followed by thawing at 30–40°C. After thawing, centrifuge at 25000–35000 rpm for 10–20 min, discard the supernatant, and... The precipitate was resuspended with lysis buffer, then frozen at -75 to -85°C for 40 to 50 minutes, thawed at 30 to 40°C, and finally centrifuged at 30,000 to 40,000 rpm for 15 to 25 minutes.

10. The use of cell membrane-encapsulated lipid nanoparticles as described in any one of claims 1 to 4 in drug delivery.

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