SPLEEN-TARGETED IONIZABLE LIPID COMPOUND, COMPOSITION COMPRISING SAME AND USE THEREOF

Abstract

The present invention provides an ionizable lipid compound, which can target spleen for delivery of biological macromolecules including nucleic acid drugs or nucleic acid vaccines with high efficiency. The present invention also relates to a lipid nanoparticle (LNP) comprising the ionizable lipid compound and an active molecule, and a pharmaceutical composition comprising the lipid nanoparticle.

Claims

1. A lipid compound of formula (I), ##STR00024## wherein R.sub.2a and R.sub.3a are each independently hydrogen, a monovalent aliphatic group, a monovalent heteroaliphatic group, a monovalent aromatic group, a monovalent heteroaromatic group, or Ht; t and s are each independently 0 or 1, and when t or s is zero, it means that the part is directly a single bond, provided that t and s are not both 0; A.sub.1, A.sub.2, and A.sub.3 are each independently a single bond, a divalent aliphatic group, a divalent heteroaliphatic group, a divalent aromatic group, or a divalent heteroaromatic group, or a combination of two of the above; each Ht is independently at each occurrence R.sub.1XR.sub.2YR.sub.3ZR.sub.4, wherein each R.sub.1 is independently at each occurrence a divalent aliphatic group, a divalent heteroaliphatic group, a divalent aromatic group, or a divalent heteroaromatic group; each X is independently at each occurrence ##STR00025## wherein m, n, p, q, and r are each independently 1-6; W is O, S, or NR.sub.c; L.sub.1, L.sub.3, L.sub.5, L.sub.7, and L.sub.9 are directly connected to R.sub.1 or R.sub.2 and are each independently a single bond, O, S, or NR.sub.d; L.sub.2, L.sub.4, L.sub.6, L.sub.8, and L.sub.10 are each independently a bond, O, S, or NR.sub.e; V is an aliphatic group, OR.sub.f, SR.sub.g, or NR.sub.hR.sub.i, wherein R.sub.b, R.sub.c, R.sub.d, R.sub.e, R.sub.f, R.sub.g, R.sub.h, and R.sub.1 are each independently hydrogen, hydroxy, an oxyaliphatic group, a monovalent aliphatic group, a monovalent heteroaliphatic group, a monovalent aromatic group, or a monovalent heteroaromatic group; Y and Z are each independently at each occurrence S or O; each R.sub.2 is independently at each occurrence a single bond, a divalent aliphatic group, a divalent heteroaliphatic group, a divalent aromatic group, or a divalent heteroaromatic group; each R.sub.3 is independently at each occurrence a single bond, a divalent aliphatic group, a divalent heteroaliphatic group, a divalent aromatic group, or a divalent heteroaromatic group; each R.sub.4 is independently at each occurrence a hydrophobic group selected from (CH.sub.2CH.sub.2O).sub.yC.sub.1-C.sub.2 alkyl, (CH.sub.2CH.sub.2O).sub.yC.sub.2 alkenyl or (CH.sub.2CH.sub.2O).sub.yC.sub.2 alkynyl, wherein y is 0 or 1 or 2.

2. The lipid compound of claim 1, wherein t and s are both 0; or t is 0 and s is 1; or t is 1 and s is 0.

3. The lipid compound of claim 1, wherein R.sub.1 is a C.sub.1-6 divalent aliphatic group or a C.sub.1-6 divalent heteroaliphatic group.

4. The lipid compound of claim 1, wherein X is ##STR00026## wherein each variable is as defined for formula (I).

5. The lipid compound of claim 4, wherein L.sub.1, L.sub.3, L.sub.5, L.sub.7, and L.sub.9 are connected to R.sub.1 and are each independently a single bond, O, S, or NH.

6. The lipid compound of claim 1, wherein X is ##STR00027## wherein R.sub.d and R.sub.e are as defined for formula (I).

7. The lipid compound of claim 6, wherein R.sup.d and R.sub.e are each independently H or a C.sub.1-4 monovalent aliphatic group.

8. The lipid compound of claim 1, wherein Y and Z are both S; or Y is S and Z is O; or Y is O and Z is S; or Y and Z are both O.

9. The lipid compound of claim 1, wherein each R.sub.2 is independently at each occurrence a single bond or a C.sub.1-6 divalent aliphatic group.

10. The lipid compound of claim 9, wherein each R.sub.2 is independently at each occurrence C.sub.1-4 divalent aliphatic group.

11. The lipid compound of claim 1, wherein each R.sub.3 is independently at each occurrence a single bond or a C.sub.1-6 divalent aliphatic group.

12. The lipid compound of claim 11, wherein each R.sub.3 is independently at each occurrence a single bond or ##STR00028## or methylene ##STR00029##

13. The lipid compound of claim 1, wherein each R.sub.4 is independently at each occurrence ethyl (C.sub.2H.sub.5), methyl (CH.sub.3), ethenyl, ethynyl, (CH.sub.2CH.sub.2O)CH.sub.3, (CH.sub.2CH.sub.2O)C.sub.2H.sub.5, (CH.sub.2CH.sub.2O)CHCH.sub.2, (CH.sub.2CH.sub.2O)CCH, (CH.sub.2CH.sub.2O).sub.2CH.sub.3, (CH.sub.2CH.sub.2O).sub.2C.sub.2H.sub.5, (CH.sub.2CH.sub.2O).sub.2CHCH.sub.2 or (CH.sub.2CH.sub.2O).sub.2CCH.

14. The lipid compound of claim 1, wherein each Ht is independently at each occurrence ##STR00030## wherein Y and Z are both S; or Y is S and Z is O; or Y is O and Z is S; or Y and Z are both O; each R.sub.3 is independently at each occurrence a single bond or ##STR00031## or methylene ##STR00032## and R.sub.44 is ethyl (C.sub.2H.sub.5), methyl (CH.sub.3), ethenyl or ethynyl.

15. The lipid compound of claim 1, wherein t is 1, s is 0, A.sub.2 is a single bond, and A.sub.1 and A.sub.3 are each independently a divalent aliphatic group or a divalent heteroaliphatic group; t is 1, s is 0, A.sub.2 is a single bond, and A.sub.1 and A.sub.3 are each independently a C.sub.1-C.sub.6 divalent aliphatic group; t is 1, s is 0, A.sub.2 is a single bond, A.sub.1 and A.sub.3 are each independently CH.sub.2CH.sub.2 or CH.sub.2CH.sub.2CH.sub.2, and R.sub.2a is hydrogen or a monovalent aliphatic group, or is hydrogen or C.sub.1-C.sub.6 alkyl or methyl.

16. The lipid compound of claim 1, wherein t is 1, s is 1, and A.sub.1, A.sub.2 and A.sub.3 are each independently a divalent aliphatic group or a divalent heteroaliphatic group; t is 1, s is 1, and A.sub.1, A.sub.2 and A.sub.3 are each independently a C.sub.1-C.sub.6 divalent aliphatic group; t is 1, s is 1, and A.sub.1, A.sub.2 and A.sub.3 are each independently CH.sub.2CH.sub.2 or CH.sub.2CH.sub.2CH.sub.2, and R.sub.2a and R.sub.3a are each independently hydrogen or a monovalent aliphatic group, or hydrogen or C.sub.1-C.sub.6 alkyl or methyl.

17. The lipid compound of claim 1, wherein is ##STR00033## wherein R.sub.2a and R.sub.3a are each independently hydrogen or a monovalent aliphatic group, or hydrogen or C.sub.1-C.sub.6 alkyl or methyl.

18. The lipid compound of claim 1, wherein the lipid compound of formula (I) is ##STR00034##

19. A pharmaceutical composition comprising a pharmaceutical carrier and a lipid nanoparticle (LNP), wherein the lipid nanoparticle comprises the lipid compound of claim 1 and a pharmaceutically active molecule.

20. The pharmaceutical composition of claim 19, wherein the pharmaceutically active molecule target the spleen as a target organ.

21. The pharmaceutical composition of claim 19, wherein the pharmaceutical active molecule is a nucleic acid, an antigen, a vaccine, an immunomodulator, or another active ingredient, or a combination thereof, that targets the spleen as a target organ.

22. The pharmaceutical composition of claim 19, wherein the LNP further comprises a phospholipid, cholesterol and a PEGylated lipid.

23. The pharmaceutical composition of claim 19, wherein the molar ratio of the lipid compound to cholesterol to phospholipid to PEGylated lipid is (about 15 to about 50):(about 38.5 to about 75):(about 10 to about 25):(about 0.5 to about 3).

24. The pharmaceutical composition of claim 19, wherein the N/P ratio of the lipid compound to the nucleic acid ranges from about 5:1 to about 20:1.

25. A method for delivering an antigen to an antigen-presenting cell of the spleen or expressing an antigen in an antigen-presenting cell of the spleen, comprising administering the pharmaceutical composition of claim 19 to a subject in need thereof.

26. The method of claim 25, wherein the antigen-presenting cell is a professional antigen-presenting cell.

27. A method for treating a disease caused by spleen damage or abnormalities, comprising administering the pharmaceutical composition of claim 19 to a subject in need thereof.

28. The method of claim 27, wherein the disease caused by spleen damage or abnormalities comprises lymphoma and leukemia.

29. The method of claim 27, wherein the pharmaceutical composition is administered systemically.

30. The method of claim 27, wherein the LNP: (i) targets the spleen or accumulates in the spleen; (ii) delivers an RNA to an antigen-presenting cell of the spleen or to an antigen-presenting cell in the spleen; and/or (iii) releases an RNA at a target organ or target tissue and/or enters a cell of the target organ or target tissue.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0086] FIG. 1 shows in vivo bioluminescence imaging images after delivery of Firefly luciferase (Fluc) mRNAs by LNPs prepared from lipid compounds YX-30, YX-32 and YX-34 (administered dose: 150 g/kg).

[0087] FIG. 2A-B shows in vivo and dissected tissue bioluminescence imaging images after delivery of Firefly luciferase (Fluc) mRNAs by LNPs prepared from lipid compound YX-32 (a), and the ratio of mRNA expression in the spleen/whole body (b).

[0088] FIG. 3 shows luciferase intensity analysis in different tissues of mice after delivery of Fluc mRNAs by different YX-32 LNP preparations.

[0089] FIG. 4 shows analysis of splenic cell uptake of YX-32/Fluc mRNA nanoparticles.

DETAILED DESCRIPTION OF EMBODIMENTS

[0090] The present invention will be further described in detail in conjunction with specific embodiments, and examples are given only to illustrate the present invention, but not to limit the scope of the present invention. The examples provided below can be used as a guide for further improvement by those of ordinary skill in the art and are not intended to limit the present invention in any way.

[0091] For all the quantitative experiments in the following examples, three repeated experiments are set up and the results are averaged.

[0092] The experimental methods in the following examples are all conventional unless otherwise specified. The materials, reagents, etc., used in the following examples can all be obtained from commercial sources unless otherwise specified.

[0093] In the following examples, cholesterol is a product from Macklin, with article number C10006595, CAS: 57-88-5.

[0094] In the following examples, both DOPE (dioleoylphosphatidylethanolamine, CAS: 4004-05-1) and DMG-PEG.sub.2000 (CAS: 147867-65-) are products from Aveto (Shanghai) Pharmaceutical Technology Co., Ltd.

[0095] The firefly luciferase mRNA in the following examples is a product from Shanghai Hongene Technology Development Co., Ltd.

[0096] In the following examples, Balb/c mice are products from Beijing Vital River Laboratory Animal Technology Co., Ltd.

Example 1: Synthesis of Ionizable Lipid Compounds

[0097] The cationizable lipid compound is synthesized from the corresponding hydrophilic amine compound and the ketal-containing acrylate TK2 by Michael addition reaction.

[0098] The hydrophobic tail molecule TK2, i.e. 2-((2-((2-(ethyloxy)ethyl)thio)propan-2-yl)thio)ethyl acrylate

##STR00020##

was synthesized in a similar manner to that in Examples 1 and 2 in CN110101665A. Specifically,

##STR00021##

2,2-(propane-2,2-diylbis(sulfanediyl))bis(ethan-1-ol) and the corresponding haloethane (e.g., bromoethane) were used as starting materials, to prepare

##STR00022##

which was then reacted with an acrylyl halide (e.g., acryloyl chloride) to obtain TK2.

[0099] .sup.1H NMR of TK2 was as follows: .sup.1H NMR (300 MHz, CDCl.sub.3) 6.41 (d, 1H), 6.12 (t, 1H), 5.83 (d, 1H), 4.32 (d, 2H), 3.59 (d, 2H), 3.46 (d, 2H), 2.92 (d, 2H), 2.82 (d, 2H), 1.61-1.38 (m, 10H), 0.89 (d, 3H).

[0100] YX-30 was synthesized from hydrophilic amine compound N,N-di(3-aminopropyl)methylamine and TK2, wherein the hydrophilic amine compound N,N-di(3-aminopropyl)methylamine and TK2 were mixed at a molar ratio of 4.3:1 and heated at 70 C. for 72 h. The crude product was purified on a silica gel column with dichloromethane/methanol as an eluent to obtain cationizable lipid compound YX-30. .sup.1H NMR of YX-30 was as follows: .sup.1H NMR (300 MHz, CDCl.sub.3) 4.22 (t, 8H), 3.59-3.50 (m, 16H), 2.86-2.77 (m, 24H), 2.45 (t, 12H), 2.28 (t, 4H), 2.18 (s, 3H), 1.60 (s, 32H), 1.20 (t, 12H).

[0101] YX-32 and YX-34 were synthesized from the corresponding hydrophilic amine compounds 2,2-diamino-N-methyldiethylamine and N,N-bis(2-aminoethyl)-N,N-dimethyl-1,3-propanediamine, respectively, and TK2, according to the above method.

[0102] YX-32: .sup.1H NMR (300 MHz, CDCl.sub.3) 4.22 (t, 8H), 3.60 (t, 8H), 3.52 (t, 8H), 2.88-2.77 (m, 24H), 2.47 (t, 8H), 1.60 (s, 24H), 1.25-1.18 (m, 20H), 1.10 (t, 12H)

[0103] YX-34: .sup.1H NMR (300 MHz, CDCl.sub.3) 4.22 (t, 8H), 3.58 (t, 8H), 3.44 (t, 8H), 2.86-2.77 (m, 24H), 2.55-2.46 (m, 16H), 2.23 (s, 3H), 1.60-1.38 (m, 26H), 1.20 (t, 12H).

##STR00023##

Example 2: Encapsulation of Firefly Luciferase mRNAs, Preparation of Lipid Nanoparticles and the Delivery Experiment

[0104] In order to achieve spleen-targeted mRNA delivery at animal level, the inventors prepared mRNA/lipid nanoparticles using the method as follows: formulating each of the cationizable lipid compounds (YX-30, YX-32 or YX-34) prepared in Example 1, cholesterol, DOPE and DMG-PEG2000 into a 10 mg/mL ethanol solution. Taking YX-32 as an example, when preparing an mRNA/LNP complex in a single time, the solution of lipid compound YX-30/YX-32/YX-34 (1.1 mg) was measured out and mixed with cholesterol, DOPE and DMG-PEG2000 in a molar ratio of 50:38.5:10:1.5, and the total volume of each solution was allowed to reach 450 L by supplementing ethanol. 150 g of firefly luciferase mRNAs were taken and added to a sodium acetate buffer (50 mM, pH=5.2), and the total volume of the solution was 1350 L. The cationizable lipid compound solution and the mRNA solution were mixed by introducing into a microfluidic machine (speed: 0.2 mL/min of ethanol solution, and 0.6 mL/min of sodium acetate buffer solution) to prepare lipid nanoparticles encapsulating mRNAs, which were dialyzed against PBS buffer for 3 hours, and the obtained samples were directly used for animal experiments. The ratio of the cationizable lipid compound YX-30/YX-32/YX-34 to mRNAs was calculated according to the molar ratio (N/P) of the nitrogen atoms in YX-30/YX-32/YX-34 to the phosphorus atoms in the phosphate skeleton of the mRNAs. In this example, N/P was 7.5.

[0105] For lipid nanoparticles encapsulating mRNAs, the concentration of RNAs therein can be detected using QUANT-IT RNA assay (Invitrogen Corporation, Carlsbad, CA), so as to evaluate for the RNA encapsulation efficiency of the nanoparticle composition. Samples were diluted in TE buffer (Solarbio, T1120, pH 8.0) to a concentration of about 5 g/mL. 50 L of the diluted sample was transferred to a polystyrene 96-well plate and 50 L of TE buffer or 50 L of 2% Triton X-100 solution (Solarbio, T8200) was added to the wells. The plate was incubated at a temperature of 37 C. for 10 minutes. Reagents were diluted 1:200 in TE buffer and 100 L of this solution was added to each well. The fluorescence intensity can be measured using a fluorescence plate reader (BIOTEK/Synergy H1) at an excitation wavelength of 480 nm and an emission wavelength of 520 nm, and the percentage of free RNAs was determined by dividing the fluorescence intensity of the intact sample (without addition of Triton X-100) by the fluorescence value of the lysed sample (caused by addition of Triton X-100).

[0106] The determination results of encapsulation efficiencies of the three LNPs for RNAs were as follows: [0107] LNPs comprising YX-30: 81.9%; [0108] LNPs comprising YX-32: 52.1% [0109] LNPs comprising YX-34: 53.9%.

[0110] In addition, the average particle size, particle dispersion index (PDI) and zeta potential of LNPs were tested by conventional methods in the art.

[0111] The test results of LNPs comprising YX-30 in this example were as follows: an average particle size of 188 nm, PDI of 0.01, and a zeta potential of 4.0 mV.

[0112] The test results of LNPs comprising YX-32 in this example were as follows: an average particle size of 195 nm, PDI of 0.03, and a zeta potential of 6.0 mV.

[0113] The test results of LNPs comprising YX-34 in this example were as follows: an average particle size of 265 nm, PDI of 0.08, and a zeta potential of 3.1 mV.

[0114] YX-30 LNPs, YX-32LNPs and YX-34 LNPs encapsulating 150 g/kg Fluc mRNAs were injected into 6-8 week-old female Balb/c mice by administration via tail vein; after 6 hours, 200 L of D-Lucifin (with a mass fraction of 30%) was injected by intraperitoneal administration, and the luciferase expression and chemiluminescence intensity in the whole body were detected by PerkinElmer IVIS Lumina III small animal in vivo optical imaging system.

[0115] In vivo imaging after delivery of Fluc mRNAs by LNPs comprising lipid compounds YX-30, YX-32 and YX-34 is shown in FIG. 1. FIG. 1 shows that the Luciferase mRNAs delivered by the three LNPs are mainly expressed at the spleen site. Among them, the targeting of the LNPs comprising YX-32 is the best, and the delivery efficiency of the LNPs comprising YX-30 is the highest, but with the slightly worse targeting than that of the LNPs comprising YX-32.

Example 3: Spleen-Targeted Delivery of mRNAs by Lipid Nanoparticles of the Present Invention

[0116] In order to verify the spleen-targeted delivery effect of the above Fluc mRNA/LNP preparations, YX-32 LNPs encapsulating Fluc mRNAs were injected into 6-8 week-old female Balb/c mice by administration via tail vein (the administered dose of mRNAs was 500 g/kg; the samples were prepared according to the method described in Example 2); after 6 hours, 200 L of D-Lucifin (with a mass fraction of 30%) was injected by intraperitoneal administration, and the luciferase expression and chemiluminescence intensity in the whole body were detected by PerkinElmer IVIS Lumina III in vivo optical imaging system for testing in mice. The mice were then sacrificed and dissected to collect tissues, and the treated heart, liver, spleen, lung, and kidney tissues were tested for luciferase expression and chemiluminescence intensity using the PerkinElmer IVIS Lumina III.

[0117] The results are shown in FIG. 2. The left panel of FIG. 2(a) shows the in vivo (i.e. the whole body) imaging image, and the right panel of FIG. 2(a) shows the tissue imaging image of each organ (including the heart, liver, spleen, lung and kidney). FIG. 2(b) shows the luminescence intensity of mRNAs in the whole body and spleen, wherein the ratio of the mRNA expression in the spleen to the mRNA expression in the whole body is as high as 90%, which fully demonstrates the targeted delivery of the LNPs of the present invention to the spleen.

Example 4: The Effect of YX-32 LNP Preparations Comprising Different Ratios of Components on Targeted mRNA Delivery

[0118] In order to further study the effect of YX-32 LNP preparations comprising different ratios of components on spleen-targeted mRNA delivery, the inventors prepared YX-32/Fluc mRNA preparations comprising different four ratios of components, and studied the expression of luciferase mRNAs at the spleen site when luciferase mRNAs were delivered by these preparations. In view of this, the YX-32 solution (1.1 mg) was measured out and mixed with cholesterol, DOPE and DMG-PEG2000 in different molar ratios (as shown in Table 1), and the total volume of the solution was allowed to reach 450 L by supplementing ethanol. The method for preparing YX-32 lipid nanoparticles encapsulating Fluc mRNAs was carried out according to the method described in Example 2. The ratio of lipid compound YX-32 to mRNAs was calculated according to the molar ratio (N/P) of the nitrogen atoms in YX-32 to the phosphorus atoms in the phosphate skeleton of the mRNAs, wherein the YX-32-01 preparation was the LNP preparation prepared in Example 2.

TABLE-US-00002 TABLE 1 YX-32 LNP preparations useful for spleen-targeted mRNA delivery N/P Sample no. ratio Lipid Cholesterol DOPE DMG-PEG2000 YX-32-01 7.5 50 38.5 10 1.5 YX-32-02 6 25 48.5 25 1.5 YX-32-03 7.5 25 48.5 25 1.5 YX-32-04 6 15 74.5 10 0.5 YX-32-05 7.5 15 74.5 10 0.5 YX-32-06 7.5 20 60 19.5 0.5 YX-32-07 7.5 25 65 9.5 0.5 YX-32-08 7.5 30 60 9.5 0.5 YX-32-09 9 25 65 9.5 0.5

[0119] In the same manner as in Example 3, 6-8 week-old female Balb/c mice were injected with each YX-32 LNP preparation encapsulating 500 g/kg of Fluc mRNAs by administration via tail vein; after 6 hours, the mice were sacrificed and dissected to collect tissues (the heart, liver, spleen, lung, and kidney) which were placed in PBS to clean the surface and weighed. 100 mg of tissues were taken and added to 500 L of formulated tissue lysis solution (IP cell lysis solution comprising 1 mM PMSF), and then the mixture was transferred to crushed ice for tissue homogenization (35000 revolutions, 10 seconds each time, repeated three times with an interval of 10 seconds between adjacent repetitions). The lysate was centrifuged at 4 C. for 15 minutes, and the supernatant was taken for later use and the protein concentration was quantified by the BCA method. 500 g of lysates from different tissues were taken, the luciferase activity in different tissues lysates was detected using the luciferase activity system (Promega, E1501) kit, and the proportions of luciferase enzyme activity in 5 different tissue lysates were calculated. FIG. 3 shows the expression level of luciferase in the spleen and the proportions of enzyme activity in the five types of tissues of heart, liver, spleen, lung and kidney as high as 65% or more, and even as high as 90% or more, after delivery of mRNAs by LNP preparations of different components.

Example 5: Preparation of YX-32/mRNA Lipid Nanoparticles Labeled with DiO Fluorescence and Cell Uptake Experiment

[0120] In order to further verify that the expression of luciferase after the administration of YX-32/mRNA lipid nanoparticles is related to the uptake of YX-32/mRNAs by spleen cells, the inventors carried out the preparation of fluorescently labeled mRNA/lipid nanoparticles and studied the cell uptake efficiency. The specific method was as follows: 13 L of DiO solution in ethanol (10 mg/mL) was added to the YX-32/Fluc mRNA lipid nanoparticles prepared in Example 1, and mixed evenly in the dark. The mixture was dialyzed using PBS as the buffer for 3 hours in the dark, and the obtained sample was directly used for the animal experiment. In order to further study the uptake efficiency of YX-32/Fluc mRNAs by spleen cells, LNPs encapsulating 500 g/kg of DiO-labeled YX-32/Fluc mRNAs were injected into 6-8 week-old female Balb/c mice by administration via tail vein; after 6 hours, the mice were sacrificed and dissected to collect the spleen tissue, and the surface of the tissue was washed in PBS. The spleen tissue was added to 250 L of digestion solution (DMEM cell culture medium containing 45 U/L of collagenase 1+25 U/L of DNAse I+30 U/L of hyaluronidase 1) and minced with a sterile blade. The minced spleen and digestion solution were transferred to a 15 mL tube, 5-10 mL of digestion solution was added, and the mixture was incubated in a constant temperature incubator at 37 C. and shaken for 1 hour. After removing, the mixture was filtered through a 70 m cell strainer, the tube and strainer were rinsed with PBS (containing 2% FBS) to make the total amount of liquid up to 10 mL. The above cell suspension was centrifuged under a condition at 4 C. for 5 minutes (speed: 300 G), and then the supernatant was discarded. The cell pellet was then resuspended with 2 mL of red blood cell lysis solution, and lysed in an ice bath for 5 minutes, during which the red blood cells were fully lysed by shaking several times. The red blood cell lysis was stopped by addition of 4 mL of PBS (containing 2% FBS), the above cell suspension was centrifuged under a condition at 4 C. for 5 minutes (speed: 300 G), and then the supernatant was discarded. The cell pellet was resuspended with 1 mL of PBS (containing 2% FBS). After resuspension, the suspension was filtered with a 70 m cell strainer, the filtrate was pipetted and blown to mix evenly, and the DiO positive ratio was detected and analyzed by a flow cytometer, that is, the cell uptake efficiency of YX-32/Fluc mRNA LNPs. The results showed that the efficiency of uptake of YX-32/mRNA lipid nanoparticles by spleen cells was about 35% or more (FIG. 4).

[0121] The above description is only preferred embodiments of the present invention; however, the scope of protection of the present invention is not limited thereto. Any changes or substitutions readily conceivable to those familiar with the technical field within the technical scope disclosed by the present invention should be covered by the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection of the claims.