Fluorine-containing polymer for spleen selective nucleic acid delivery, polymer lipid hybrid nano-particles and preparation method and application of polymer lipid hybrid nano-particles

Abstract

The invention provides a fluorine-containing polymer for spleen selective nucleic acid delivery, a polymer lipid hybrid nanoparticle and a preparation method and application of the polymer lipid hybrid nanoparticle. The preparation method of the fluorine-containing polymer for spleen selective nucleic acid delivery comprises the step of carrying out ring-opening addition reaction on alkylene oxide, fluorine-substituted alkylene oxide and polyethyleneimine to prepare the fluorine-containing polymer. The preparation method of the fluorine-containing polymer is simple, mild in condition and high in finished product yield; the obtained fluorine-containing polymer is used for preparing polymer lipid hybrid nanoparticles, nucleic acid can be specifically delivered to the spleen and plays a role, and the fluorine-containing polymer has a good transfection effect on the dendritic cells of the spleen and also has good biological safety.

Description

Technical Field

[0001] The invention relates to a fluorine-containing polymer and polymer-lipid hybrid nanoparticle for spleen selective nucleic acid delivery and a preparation method and application thereof, belonging to the technical field of biomedicine. Background Art

[0002] In 2018, the FDA approved the first lipid / lipidoid nanoparticle (LNP)-based siRNA drug (Onpattro); two years later, two SARSCoV-2 vaccines based on LNPs containing mRNA (Comirnaty, Spikevax) also entered the clinic, saving millions of lives during the COVID-19 pandemic. Nucleic acid therapy is also gaining more and more attention from researchers.

[0003] RNA therapy includes antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), microRNAs (miRNAs), messenger RNAs (mRNAs), and single guide RNAs (sgRNAs)-mediated CRISPR-Cas9 systems, which can manipulate essentially any gene through different modes of action. However, RNA therapeutics are susceptible to nucleases and cannot penetrate cells due to their large size and negative charge. Current nucleic acid delivery vectors mainly include viral vectors, LNPs, polymer vectors, micelles, vesicle vectors, etc. LNPs or polymer vectors are usually composed of four components: ionizable lipids (or amine-rich polymer structures), phospholipids, cholesterol, and PEGylated lipids, among which ionizable lipids play a major role in protecting RNA and promoting its cytoplasmic transport. Ionizable lipids are positively charged at acidic pH to condense RNA into LNPs, but are neutral at physiological pH to reduce toxicity. After cellular uptake, they can be protonated in acidic endosomes and interact with anionic phospholipids in vivo to form conical ion pairs that are incompatible with the bilayer. These cationic anionic lipids are able to drive the transition from the bilayer structure to the inverted hexagonal HII phase, thereby promoting membrane fusion / disruption, endosomal escape, and cargo release into the cytoplasm.

[0004] The existing delivery vectors for RNA delivery still have many shortcomings, including polar immune response and long-term toxicity caused by ionizable lipids in LNPs, and cumbersome synthesis process. At the same time, extrahepatic delivery is a major challenge for the widespread application of RNA therapy, and selective delivery of nucleic acids to the spleen is even more challenging. In the prior art, there are reports of delivery vectors that selectively deliver nucleic acids to the lungs, but they cannot effectively and selectively deliver nucleic acids to the spleen, which will seriously restrict the nucleic acid treatment of spleen-related diseases.

[0005] Therefore, the rational design of ionizable lipids with targeting functions, which are simple to synthesize and have low toxicity, and can effectively and selectively deliver nucleic acids to the spleen for nucleic acid therapy of spleen-related diseases, has important research value and broad application prospects. Summary of the invention

[0006] In order to overcome the deficiencies of the prior art, the present invention provides a fluorinated polymer, polymer lipid hybrid nanoparticles for spleen selective nucleic acid delivery, and a preparation method and application thereof. The fluorinated polymer of the present invention is prepared by ring-opening addition of polyethyleneimine, alkylene oxide, and fluorine-substituted alkylene oxide; the preparation method is simple, the conditions are mild, and the yield of the finished product is high; the obtained fluorinated polymer is used to prepare polymer lipid hybrid nanoparticles, which can specifically deliver nucleic acids to the spleen and exert their effects, have a good transfection effect on splenic dendritic cells, and have good biological safety.

[0007] The technical solution adopted by the present invention is as follows:

[0008] A method for preparing a fluorine-containing polymer for spleen selective nucleic acid delivery comprises the steps of preparing the fluorine-containing polymer through a ring-opening addition reaction of alkylene oxide, fluorine-substituted alkylene oxide and polyethylene imine.

[0009] Preferably, according to the present invention, the polyethyleneimine (PEI) is branched polyethyleneimine (BPEI) or linear polyethyleneimine (LPEI); preferably, the polyethyleneimine (PEI) is branched polyethyleneimine (BPEI).

[0010] Preferably according to the present invention, the weight average molecular weight Mw of polyethyleneimine (PEI) is 300-25000.

[0011] Preferably according to the present invention, the molar ratio of polyethyleneimine (PEI) to the total molar ratio of alkylene oxide and fluorine-substituted alkylene oxide is 1:6-1:14; preferably, the molar ratio of polyethyleneimine (PEI) to the total molar ratio of alkylene oxide and fluorine-substituted alkylene oxide is 1:9-1:13; most preferably, the molar ratio of polyethyleneimine (PEI) to the total molar ratio of alkylene oxide and fluorine-substituted alkylene oxide is 1:11.

[0012] Preferably, according to the present invention, the molar ratio of alkylene oxide to fluorine-substituted alkylene oxide is 20:80-80:20; preferably, the molar ratio of alkylene oxide to fluorine-substituted alkylene oxide is 20:80-40:60; most preferably, the molar ratio of alkylene oxide to fluorine-substituted alkylene oxide is 40:60.

[0013] According to the present invention, preferably, the hydrocarbon group in the alkylene oxide is selected from a saturated, unbranched, non-halogenated aliphatic hydrocarbon group with carbon atoms of C1 to C20; preferably, the hydrocarbon group in the alkylene oxide is selected from a saturated, unbranched, non-halogenated aliphatic hydrocarbon group with carbon atoms of C4 to C18; more preferably, the alkylene oxide is selected from one of 1,2-epoxyhexane, 1,2-epoxyoctane, 1,2-epoxydecane, 1,2-epoxydodecane or 1,2-epoxytetradecane.

[0014] According to the present invention, the hydrocarbon group in the fluorinated alkylene oxide is preferably selected from a saturated, unbranched, perfluorinated aliphatic hydrocarbon group having carbon atoms of C1-C20; preferably, the hydrocarbon group in the fluorinated alkylene oxide is selected from a saturated, unbranched, perfluorinated aliphatic hydrocarbon group having carbon atoms of C4-C18; more preferably, the fluorinated alkylene oxide is selected from one of 2,2,3,3,4,4,5,5,6,6,7,7,7-tridecafluoroheptyl oxirane (F9), 3-(perfluoro-n-octyl)-1,2-epoxypropane (F11) or 1H,2H,3H,3H-perfluoro-1,2-epoxytridecane (F13).

[0015] Preferably, according to the present invention, the ring-opening addition reaction temperature is 70-100° C., and the ring-opening addition reaction time is 36-75 hours; preferably, the ring-opening addition reaction temperature is 85-90° C., and the ring-opening addition reaction time is 48-60 hours.

[0016] Preferably according to the present invention, the ring-opening addition reaction is carried out in a light-proof environment with stirring.

[0017] According to a preferred embodiment of the present invention, the post-treatment method of the product obtained by the ring-opening addition reaction is as follows: the product is dialyzed with anhydrous ethanol and then dried.

[0018] A fluorine-containing polymer for spleen-selective nucleic acid delivery is prepared by the above method.

[0019] A polymer lipid hybrid nanoparticle comprises the following raw materials in percentage by mass: 20-80% of fluorine-containing polymer, 2-40% of polyethylene glycolated lipid, 5-60% of steroid and 0-10% of auxiliary lipid.

[0020] Preferably according to the present invention, the PEGylated lipid is selected from one of distearoylphosphatidylethanolamine-polyethylene glycol 2000, 1,2-dimyristoyl-rac-glycerol-3-methoxy-polyethylene glycol 2000, d-α succinic acid tocopheryl polyethylene glycol ester, 2-myristyl stannanoyl-3-phosphatidylethanolamine-polyethylene glycol 2000, dipalmitoylphosphatidylethanolamine-polyethylene glycol 2000 or 1,2-distearoyl-rac-glycerol-polyethylene glycol 2000; preferably, the PEGylated lipid is one of distearoylphosphatidylethanolamine-polyethylene glycol 2000 or 1,2-dimyristoyl-rac-glycerol-3-methoxy-polyethylene glycol 2000.

[0021] Preferably according to the present invention, the steroid is selected from one of cholesterol, β-sitosterol, stigmasterol, sitosterol, stigmasterol, sitostanol, ergosterol, cholestanol, dehydrocholesterol, dihydrocholesterol, hydroxycholesterol or saponasterol; preferably, the steroid is selected from one of cholesterol, β-sitosterol, stigmasterol, stigmasterol, ergosterol or saponasterol; most preferably, the steroid is cholesterol.

[0022] Preferably according to the present invention, the auxiliary lipid is selected from 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-SN-glycero-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dimyristoyl-sn-glycero-3-phospho-rac-(1-glycerol) (DPPG), 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine (DSPE), 1,2-dipalmitoyl-rac-glycero-3-phosphocholine (DPPC), dimyristoylphosphatidylcholine (DMPC) or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC); preferably, the auxiliary lipid is DOPE.

[0023] The method for preparing the polymer lipid hybrid nanoparticles comprises the steps of:

[0024] ① Dissolving the fluorinated polymer, PEGylated lipid, steroid, and auxiliary lipid in an organic solvent to obtain a premixed solution;

[0025] ② Add the premixed solution dropwise into the buffer solution under vortex conditions to obtain polymer lipid hybrid nanoparticles.

[0026] Preferably according to the present invention, in step ①, the organic solvent is anhydrous ethanol or anhydrous methanol; preferably, the organic solvent is anhydrous ethanol; in the premix, the mass concentration of the fluorinated polymer is 2-200 mg / mL; preferably, in the premix, the mass concentration of the fluorinated polymer is 100 mg / mL.

[0027] Preferably according to the present invention, the buffer is acetic acid-sodium acetate buffer or citric acid-sodium citrate buffer; the buffer pH is 4-8; preferably, the buffer is acetic acid-sodium acetate buffer; the buffer pH is 4-6; most preferably, the buffer pH is 5.5.

[0028] Preferably according to the present invention, the volume ratio of the premix solution to the buffer solution is 1:8 to 1:100; preferably, the volume ratio of the premix solution to the buffer solution is 1:30.

[0029] Preferably, according to the present invention, the premixed solution is added dropwise into the buffer solution under vortex conditions to obtain a mixed solution, which is a polymer lipid hybrid nanoparticle dispersion and can be used directly later; alternatively, the obtained mixed solution is dried to obtain polymer lipid hybrid nanoparticles.

[0030] The above-mentioned fluorinated polymer or polymer lipid hybrid nanoparticles are used for selective delivery of nucleic acid drugs to the spleen.

[0031] Preferably according to the present invention, the fluorinated polymer or polymer lipid hybrid nanoparticles are used for selective delivery of nucleic acid drugs to splenic dendritic cells.

[0032] Preferably according to the present invention, the nucleic acid drug is selected from one or a combination of two or more of mRNA, small interfering RNA (siRNA), microRNA (microRNA), antisense oligonucleotide (ASO), circular RNA (circRNA), short hairpin RNA (shRNA), DNA or nucleic acid aptamers.

[0033] According to the preferred embodiment of the present invention, the specific application method is as follows:

[0034] ① Adding the nucleic acid drug to the above polymer lipid hybrid nanoparticle dispersion, mixing thoroughly, and standing at room temperature to obtain a polymer-lipid hybrid nanoparticle-nucleic acid complex;

[0035] ② The polymer-lipid hybrid nanoparticle-nucleic acid complex is administered to an organism to achieve spleen-selective delivery of nucleic acid drugs in the organism.

[0036] Preferably, the mass ratio of the polymer lipid hybrid nanoparticle to the nucleic acid drug is 2:1-30:1; preferably, the mass ratio of the polymer lipid hybrid nanoparticle to the nucleic acid drug is 5:1-20:1; further preferably, the mass ratio of the polymer lipid hybrid nanoparticle to the nucleic acid drug is 10:1-20:1; most preferably, the mass ratio of the polymer lipid hybrid nanoparticle to the nucleic acid drug is 15:1.

[0037] Preferably, the standing time at room temperature is 5-60 minutes; preferably, the standing time at room temperature is 10-30 minutes.

[0038] Preferably, the polymer-lipid hybrid nanoparticle-nucleic acid complex is administered to an organism by a method including intravenous injection, intramuscular injection, tracheal administration, subcutaneous injection, nasal inhalation or oral administration.

[0039] The technical features and beneficial effects of the present invention are as follows:

[0040] (1) The raw materials of the present invention are easily available, the preparation process is mild, simple, safe, and the yield of the finished product is high.

[0041] (2) The present invention introduces fluorinated alkylene oxide segments into polymers of alkylene oxide and polyethyleneimine, so that the polymer lipid hybrid nanoparticles prepared by the present invention can specifically deliver nucleic acid drugs to the spleen dendritic cells of the organism and exert their effects, with excellent transfection effect and good biosafety. The addition of fluorinated alkylene oxides can enhance the cellular uptake of nanoparticles in vivo and in vitro, enhance endosomal escape, and thus enhance transfection; secondly, the addition of fluorinated alkylene oxides will reduce the surface potential of nanoparticles and increase the particle size of nanoparticles, which may be related to organ-selective spleen delivery; finally, the addition of fluorinated alkylene oxides will change the adsorption type of the protein corona on the surface of nanoparticles, which will change the organ selectivity of nanoparticles.

[0042] (3) The construction of the spleen dendritic cell targeted nucleic acid drug delivery system of the present invention can achieve efficient transfection of spleen dendritic cells without adding active targeting molecules or auxiliary components, effectively reducing the application cost.

[0043] (4) The targeted delivery of splenic dendritic cells of the present invention provides a possibility and guidance for achieving immunotherapy with nucleic acid drugs.

[0044] (5) The polymer lipid hybrid nanoparticles prepared by the present invention have good storage stability and can be stored for a long time in a relatively mild environment.

[0045] (6) In the preparation method of the polymer of the present invention, if no alkylene oxide is added and only fluorine-substituted alkylene oxide is used, the prepared nanoparticles have poor dispersibility and cannot achieve the delivery of nucleic acid drugs. If the molar ratio of alkylene oxide to fluorine-substituted alkylene oxide is not appropriate, the selective delivery and transfection effect of the nanoparticles will be reduced. If the type of fluorine-substituted alkylene oxide or the type of alkylene oxide is not appropriate, the selective delivery and transfection effect of the nanoparticles will also be reduced. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 The hydrated particle size (a), zeta potential (b), pKa (c), and TEM image (d) of polymer-lipid hybrid nanoparticles and polymer-lipid hybrid nanoparticle-nucleic acid complexes.

[0047] Figure 2 Figure 2 shows the mRNA encapsulation efficiency of polymer-lipid hybrid nanoparticles (a) and their cytotoxicity to HeLa cells (b).

[0048] Figure 3 The expression effect of polymer-lipid hybrid nanoparticles loaded with EGFP mRNA (mEGFP) in HeLa cells (a, b) and fluorescence microscopy images (c).

[0049] Figure 4 Bioluminescence imaging of mice in vivo and in vitro 6 hours after intravenous injection of polymer-lipid hybrid nanoparticles loaded with mRNA expressing firefly luciferase (mLuc).

[0050] Figure 5 This is a quantitative analysis of bioluminescence in the heart, liver, spleen, lung, and kidney of mice 6 hours after intravenous injection of polymer-lipid hybrid nanoparticles loaded with mRNA expressing firefly luciferase (mLuc) into mice.

[0051] Figure 6 Pseudo-color density map of flow cytometry for polymer-lipid hybrid nanoparticle-mediated mRNA delivery to spleen cell subsets (a) and quantitative analysis of the proportion of DiO-positive cells in the main spleen cell subsets (b).

[0052] Figure 7 Histopathology report of heart, liver, spleen, lung, and kidney in mice 24 hours after intravenous injection of polymer-lipid hybrid nanoparticles loaded with mRNA. DETAILED DESCRIPTION

[0053] The technical scheme of the present invention is described clearly and completely below. Obviously, the described embodiments are only some embodiments of the present invention, rather than all embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention. The experimental methods used in the embodiments are conventional methods unless otherwise specified. The materials, reagents, etc. used in the embodiments, unless otherwise specified, can be obtained from commercial sources.

[0054] In the embodiment, the polyethyleneimine is branched polyethyleneimine with a weight average molecular weight Mw of 600.

[0055] Example 1

[0056] A method for preparing a fluorinated polymer for spleen selective nucleic acid delivery comprises the steps of:

[0057] 1,2-epoxydecane and 2,2,3,3,4,4,5,5,6,6,7,7,7-tridecafluoroheptyl oxirane are mixed evenly at a molar ratio of 80:20 to obtain an oxirane premix; polyethyleneimine is added and mixed evenly, wherein the molar ratio of polyethyleneimine to the total molar ratio of 1,2-epoxydecane and tridecafluoroheptyl oxirane is 1:11; stirring is continued at 90°C for 60 hours under light-proof conditions. The obtained crude product is dialyzed with anhydrous ethanol and then dried at 50°C to remove ethanol to obtain a fluorinated polymer.

[0058] Example 2

[0059] A method for preparing a fluorinated polymer for spleen selective nucleic acid delivery comprises the steps of:

[0060] 1,2-epoxydecane and 2,2,3,3,4,4,5,5,6,6,7,7,7-tridecafluoroheptyl oxirane are mixed evenly at a molar ratio of 40:60 to obtain an oxirane premix; polyethyleneimine is added and mixed evenly, wherein the molar ratio of polyethyleneimine to the total molar ratio of 1,2-epoxydecane and tridecafluoroheptyl oxirane is 1:11; stirring is continued at 90°C for 60 hours under light-proof conditions. The obtained crude product is dialyzed with anhydrous ethanol and then dried at 50°C to remove ethanol to obtain a fluorinated polymer.

[0061] Example 3

[0062] A method for preparing a fluorinated polymer for spleen selective nucleic acid delivery comprises the steps of:

[0063] 1,2-epoxydecane and 2,2,3,3,4,4,5,5,6,6,7,7,7-tridecafluoroheptyl oxirane are mixed evenly at a molar ratio of 20:80 to obtain an oxirane premix; polyethyleneimine is added and mixed evenly, wherein the molar ratio of polyethyleneimine to the total molar ratio of 1,2-epoxydecane and tridecafluoroheptyl oxirane is 1:11; stirring is continued at 90°C for 60 hours under light-proof conditions. The obtained crude product is dialyzed with anhydrous ethanol and then dried at 50°C to remove ethanol to obtain a fluorinated polymer.

[0064] Example 4

[0065] A method for preparing a fluorinated polymer for spleen selective nucleic acid delivery is as described in Example 2, except that 2,2,3,3,4,4,5,5,6,6,7,7,7-tridecafluoroheptyl oxirane is replaced by 3-(perfluoro-n-octyl)-1,2-propylene oxide; the other steps and conditions are the same as in Example 2.

[0066] Comparative Example 1

[0067] A method for preparing a polymer comprises the steps of:

[0068] Polyethyleneimine and 1,2-epoxydecane were mixed uniformly at a molar ratio of 1:11, and stirred continuously at 90° C. for 60 hours under light-proof conditions. The obtained crude product was dialyzed with anhydrous ethanol and then dried at 50° C. to remove ethanol, thereby obtaining a polymer.

[0069] Comparative Example 2

[0070] A method for preparing a polymer comprises the steps of:

[0071] Polyethyleneimine and 2,2,3,3,4,4,5,5,6,6,7,7,7-tridecafluoroheptyl oxirane were mixed uniformly at a molar ratio of 1:11, and stirred continuously at 90° C. for 60 hours under light-proof conditions. The obtained crude product was dialyzed with anhydrous ethanol and then dried at 50° C. to remove ethanol, thereby obtaining a polymer.

[0072] Example 5

[0073] A polymer lipid hybrid nanoparticle comprises the following raw materials in percentage by mass: 57% of a fluorine-containing polymer (prepared in Example 1), 28.5% of a pegylated lipid (1,2-dimyristoyl-rac-glycerol-3-methoxy-polyethylene glycol 2000, DMG-PEG), and 14.5% of a steroid (cholesterol).

[0074] The preparation method of the polymer lipid hybrid nanoparticles comprises the following steps:

[0075] Fluoropolymer, DMG-PEG and cholesterol were dissolved in anhydrous ethanol to obtain a lipid premix (wherein the mass concentration of the fluorine-containing polymer was 100 mg / mL). The lipid premix was added dropwise into an acetic acid-sodium acetate buffer solution with a pH of 5.5 under vortex conditions (the volume ratio of the lipid premix solution to the buffer solution was 1:30) to obtain a polymer lipid hybrid nanoparticle dispersion, which was named PEI-F20.

[0076] Example 6

[0077] A polymer lipid hybrid nanoparticle, as described in Example 5, except that the fluorine-containing polymer prepared in Example 1 is replaced by the fluorine-containing polymer prepared in Example 2; and the other raw material compositions are the same as in Example 5.

[0078] The preparation method of the polymer lipid hybrid nanoparticles is as described in Example 5, except for the above differences. The obtained polymer lipid hybrid nanoparticle dispersion is named PEI-F60.

[0079] Example 7

[0080] A polymer lipid hybrid nanoparticle, as described in Example 5, except that the fluorine-containing polymer prepared in Example 1 is replaced by the fluorine-containing polymer prepared in Example 3; the other raw material compositions are the same as in Example 5.

[0081] The preparation method of the polymer lipid hybrid nanoparticles is as described in Example 5, except for the above differences. The obtained polymer lipid hybrid nanoparticle dispersion is named PEI-F80.

[0082] Example 8

[0083] A polymer lipid hybrid nanoparticle, as described in Example 5, except that the fluorine-containing polymer prepared in Example 1 is replaced by the fluorine-containing polymer prepared in Example 4; the other raw material compositions are the same as in Example 5.

[0084] The preparation method of the above polymer lipid hybrid nanoparticles is as described in Example 5, with the same differences as above.

[0085] Comparative Example 3

[0086] A polymer lipid hybrid nanoparticle, as described in Example 5, except that the fluorine-containing polymer prepared in Example 1 is replaced by the polymer prepared in Comparative Example 1; and the other raw material compositions are the same as in Example 5.

[0087] The preparation method of the polymer lipid hybrid nanoparticles is as described in Example 5, except for the above differences. The obtained polymer lipid hybrid nanoparticle dispersion is named PEI-H.

[0088] Comparative Example 4

[0089] A polymer lipid hybrid nanoparticle, as described in Example 5, except that the fluorine-containing polymer prepared in Example 1 is replaced by the fluorine-containing polymer prepared in Comparative Example 2; and the other raw material compositions are the same as in Example 5.

[0090] The preparation method of the polymer lipid hybrid nanoparticles is as described in Example 5, except for the above differences. The obtained polymer lipid hybrid nanoparticle dispersion is named PEI-F100.

[0091] Application Example 1

[0092] An application of polymer lipid hybrid nanoparticles, the application method is as follows:

[0093] The polymer lipid hybrid nanoparticle dispersion prepared in Example 5 was quickly mixed with mRNA, wherein the mass ratio of polymer lipid hybrid nanoparticle to mRNA was 5:1, 10:1, 15:1 and 20:1 respectively; and left at room temperature for 20 minutes to obtain a polymer-lipid hybrid nanoparticle-nucleic acid complex. Among them, the sample with a mass ratio of polymer lipid hybrid nanoparticle to mRNA of 15:1 was named PEI-F20 / mRNA.

[0094] Application Example 2

[0095] An application of polymer lipid hybrid nanoparticles is as described in Application Example 1, except that the polymer lipid hybrid nanoparticle dispersion prepared in Example 5 is replaced by the polymer lipid hybrid nanoparticle dispersion prepared in Example 6; the other steps and conditions are the same as those in Application Example 1. The sample with a mass ratio of polymer lipid hybrid nanoparticles to mRNA of 15:1 is named PEI-F60 / mRNA.

[0096] Application Example 3

[0097] An application of polymer lipid hybrid nanoparticles is as described in Application Example 1, except that: the polymer lipid hybrid nanoparticle dispersion prepared in Example 5 is replaced by the polymer lipid hybrid nanoparticle dispersion prepared in Example 7; the other steps and conditions are the same as those in Application Example 1. The sample with a mass ratio of polymer lipid hybrid nanoparticles to mRNA of 15:1 is named PEI-F80 / mRNA.

[0098] Application Example 4

[0099] An application of polymer lipid hybrid nanoparticles is as described in Application Example 1, except that the polymer lipid hybrid nanoparticle dispersion prepared in Example 5 is replaced by the polymer lipid hybrid nanoparticle dispersion prepared in Example 8; the other steps and conditions are the same as in Application Example 1. The mass ratio of polymer lipid hybrid nanoparticles to mRNA is 15:1.

[0100] Application Comparative Example 5

[0101] An application of polymer lipid hybrid nanoparticles is as described in Application Example 1, except that: the polymer lipid hybrid nanoparticle dispersion prepared in Example 5 is replaced by the polymer lipid hybrid nanoparticle dispersion prepared in Comparative Example 3; the other steps and conditions are the same as those in Application Example 1. Among them, the sample with a mass ratio of polymer lipid hybrid nanoparticles to mRNA of 15:1 is named PEI-H / mRNA.

[0102] Application Comparative Example 6

[0103] An application of polymer lipid hybrid nanoparticles is as described in Application Example 1, except that: the polymer lipid hybrid nanoparticle dispersion prepared in Example 5 is replaced by the polymer lipid hybrid nanoparticle dispersion prepared in Comparative Example 4; the other steps and conditions are the same as those in Application Example 1. Among them, the sample with a mass ratio of polymer lipid hybrid nanoparticles to mRNA of 15:1 is named PEI-F100 / mRNA.

[0104] Test Example 1

[0105] The polymer-lipid hybrid nanoparticles prepared in Examples 5-7 and Comparative Example 3 and the polymer-lipid hybrid nanoparticle-nucleic acid complexes prepared in Application Examples 1-3 and Comparative Example 5 were subjected to corresponding physicochemical characterizations, such as Figure 1 As shown, the obtained nanoparticles are all spherical, with a hydrated particle size of about 150-250 nm, a positive zeta potential, and a pKa between 7 and 8.2. Figure 1 d is the TEM image of PEI-F60 / mRNA.

[0106] Test Example 2

[0107] The mRNA encapsulation efficiency of the nanoparticles in the polymer-lipid hybrid nanoparticle-nucleic acid complex prepared in Examples 1-3 and Comparative Example 5 was quantitatively analyzed.

[0108] like Figure 2 As shown in (a), the encapsulation efficiency of mRNA by PEI-H, PEI-F20, and PEI-F60 all reached more than 80%, and the encapsulation efficiency of PEI-F80 dropped sharply to about 50% when the mass ratio was 5:1.

[0109] The polymer-lipid hybrid nanoparticle-nucleic acid complex prepared in Application Example 2 and Comparative Application Example 5 (wherein the mass ratio of polymer-lipid hybrid nanoparticle to mRNA is 15:1) was used to treat HeLa cells at mRNA concentrations of 2 μg / ml, 4 μg / ml, and 8 μg / ml to examine the cytotoxicity of the complex. Figure 2 As shown in (b), there was basically no cytotoxicity when the mRNA concentration was lower than 4 μg / ml.

[0110] Test Example 3

[0111] The polymer-lipid hybrid nanoparticle-nucleic acid complex prepared in Application Examples 1-4, Application Comparative Example 5 and Application Comparative Example 6 (wherein mRNA was replaced with mEGFP, and the mass ratio of polymer-lipid hybrid nanoparticle to mEGFP was 15:1) was used to treat HeLa cells at a mEGFP concentration of 0.4 μg / ml. The cells were cultured at 37°C and 5% CO2 for 24 hours, and the expression of EGFP in HeLa cells was observed by fluorescence microscopy, and quantitative analysis was performed by flow cytometry.

[0112] like Figure 3 As shown, PEI-F60 has the best transfection efficiency. The transfection efficiency of the polymer-lipid hybrid nanoparticle-nucleic acid complex prepared in Application Example 4 in HeLa cells is 0.

[0113] Test Example 4

[0114] The polymer-lipid hybrid nanoparticle-nucleic acid complex prepared in Application Example 2 and Comparative Application Example 5 (wherein mRNA was replaced by mLuc, and the mass ratio of polymer-lipid hybrid nanoparticle to mLuc was 15:1) was intravenously injected into mice at a mLuc dose of 0.2 μg / g. Six hours after intravenous injection, the expression of mLuc in the mice was observed using a small animal imaging device.

[0115] like Figure 4 , 5 As shown, the organ distribution of mLuc in mice was analyzed by in vitro imaging and quantitative analysis of bioluminescent signals in the heart, liver, spleen, lung and kidney of mice. PEI-F60 has a good mLuc expression effect in the spleen of mice.

[0116] Test Example 5

[0117] The polymer-lipid hybrid nanoparticle-nucleic acid complex prepared in Application Example 2 and Comparative Example 5 (wherein the mass ratio of polymer-lipid hybrid nanoparticle to mRNA was 15:1) was intravenously injected into mice at a dose of 0.2 μg / g of mRNA. Six hours after intravenous injection, the cell selectivity of PEI-H and PEI-F60 for mRNA spleen targeted delivery was investigated. The results are shown in Figure 6 As shown, PEI-H and PEI-F60 labeled with DIO were used to deliver mouse spleen cell populations, and different types of spleen cells were immunophenotyped by corresponding markers or antigens, including macrophages (CD45+), epithelial cells (CD326+), endothelial cells (CD31+), and dendritic cells (CD11c+). Using fluorescence activated cell sorting analysis, it was found that about 30% of dendritic cells in the RNA delivered by PEI-F60 could be labeled, showing the potential for immunotherapy.

[0118] The polymer-lipid hybrid nanoparticle-nucleic acid complex prepared in Application Example 2 and Comparative Application Example 5 (wherein the mass ratio of polymer-lipid hybrid nanoparticle to mRNA was 15:1) was intravenously injected into mice at a dose of 0.2 μg / g of mRNA. After 24 hours, the heart, liver, spleen, lung, and kidney of the mice were stained with H&E for pathological examination. Figure 7 As shown, compared with untreated mice, the organs of mice treated with the complex showed no obvious lesions, indicating that the polymer-lipid hybrid nanoparticles of the present invention have good biosafety in vivo at a dose that achieves effective transfection.

Claims

1. A method for preparing a fluorine-containing polymer for spleen-selective nucleic acid delivery, comprising the steps of: preparing a fluorine-containing polymer by a ring-opening addition reaction of an alkylene oxide, a fluorine-substituted alkylene oxide and polyethyleneimine.

2. The method for preparing a fluorinated polymer for spleen selective nucleic acid delivery according to claim 1, characterized in that: Includes one or more of the following conditions: i. The polyethyleneimine (PEI) is branched polyethyleneimine (BPEI) or linear polyethyleneimine (LPEI); preferably, the polyethyleneimine (PEI) is branched polyethyleneimine (BPEI); ii. Polyethyleneimine (PEI) weight average molecular weight Mw is 300-25000; iii. The molar ratio of polyethyleneimine (PEI) to the total molar ratio of alkylene oxide and fluorine-substituted alkylene oxide is 1:6-1:14; preferably, the molar ratio of polyethyleneimine (PEI) to the total molar ratio of alkylene oxide and fluorine-substituted alkylene oxide is 1:9-1:13; most preferably, the molar ratio of polyethyleneimine (PEI) to the total molar ratio of alkylene oxide and fluorine-substituted alkylene oxide is 1:11; iv. The hydrocarbon group in the alkylene oxide is selected from a saturated, unbranched, non-halogenated aliphatic hydrocarbon group having carbon atoms of C1 to C20; preferably, the hydrocarbon group in the alkylene oxide is selected from a saturated, unbranched, non-halogenated aliphatic hydrocarbon group having carbon atoms of C4 to C18; more preferably, the alkylene oxide is selected from one of 1,2-epoxyhexane, 1,2-epoxyoctane, 1,2-epoxydecane, 1,2-epoxydodecane or 1,2-epoxytetradecane; v. The ring-opening addition reaction temperature is 70-100° C., and the ring-opening addition reaction time is 36-75 hours; preferably, the ring-opening addition reaction temperature is 85-90° C., and the ring-opening addition reaction time is 48-60 hours; vi. The ring-opening addition reaction is carried out in a light-proof environment with stirring.

3. The method for preparing a fluorinated polymer for spleen selective nucleic acid delivery according to claim 1, characterized in that: The molar ratio of alkylene oxide to fluorine-substituted alkylene oxide is 20:80-80:20; preferably, the molar ratio of alkylene oxide to fluorine-substituted alkylene oxide is 20:80-40:60; most preferably, the molar ratio of alkylene oxide to fluorine-substituted alkylene oxide is 40:

60.

4. The method for preparing a fluorinated polymer for spleen selective nucleic acid delivery according to claim 1, characterized in that: The hydrocarbon group in the fluorinated alkylene oxide is selected from a saturated, unbranched, perfluorinated aliphatic hydrocarbon group with carbon atoms of C1-C20; preferably, the hydrocarbon group in the fluorinated alkylene oxide is selected from a saturated, unbranched, perfluorinated aliphatic hydrocarbon group with carbon atoms of C4-C18; more preferably, the fluorinated alkylene oxide is selected from one of 2,2,3,3,4,4,5,5,6,6,7,7,7-tridecafluoroheptyl oxirane (F9), 3-(perfluoro-n-octyl)-1,2-epoxypropane (F11) or 1H,2H,3H,3H-perfluoro-1,2-epoxytridecane (F13).

5. A fluorinated polymer for spleen selective nucleic acid delivery, characterized in that Prepared by the method according to any one of claims 1 to 4.

6. A polymer lipid hybrid nanoparticle, characterized in that: The raw material composition comprises the following mass percentages: 20-80% of fluorine-containing polymer, 2-40% of polyethylene glycol lipid, 5-60% of steroid and 0-10% of auxiliary lipid.

7. The polymer-lipid hybrid nanoparticle according to claim 6, characterized in that: Includes one or more of the following conditions: i. The PEGylated lipid is selected from one of distearoylphosphatidylethanolamine-polyethylene glycol 2000, 1,2-dimyristoyl-rac-glycerol-3-methoxy-polyethylene glycol 2000, d-α succinic acid tocopheryl polyethylene glycol ester, 2-myristyl stannoyl-3-phosphatidylethanolamine-polyethylene glycol 2000, dipalmitoylphosphatidylethanolamine-polyethylene glycol 2000 or 1,2-distearoyl-rac-glycerol-polyethylene glycol 2000; preferably, the PEGylated lipid is one of distearoylphosphatidylethanolamine-polyethylene glycol 2000 or 1,2-dimyristoyl-rac-glycerol-3-methoxy-polyethylene glycol 2000; ii. The steroid is selected from one of cholesterol, β-sitosterol, stigmasterol, sitosterol, stigmasterol, sitostanol, ergosterol, cholestanol, dehydrocholesterol, dihydrocholesterol, hydroxycholesterol or saponasterol; preferably, the steroid is selected from one of cholesterol, β-sitosterol, stigmasterol, stigmasterol, ergosterol or saponasterol; most preferably, the steroid is cholesterol; iii. The auxiliary lipid is selected from 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-SN-glycero-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dimyristoyl-sn-glycero-3-phospho-rac-(1-glycerol) (DPPG), 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine (DSPE), 1,2-dipalmitoyl-rac-glycero-3-phosphocholine (DPPC), dimyristoylphosphatidylcholine (DMPC) or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC); preferably, the auxiliary lipid is DOPE.

8. A method for preparing polymer lipid hybrid nanoparticles according to any one of claims 6 to 7, comprising the steps of: ① Dissolving the fluorinated polymer, PEGylated lipid, steroid, and auxiliary lipid in an organic solvent to obtain a premixed solution; ② Add the premixed solution dropwise into the buffer solution under vortex conditions to obtain polymer lipid hybrid nanoparticles; Preferably, in step ①, the organic solvent is anhydrous ethanol or anhydrous methanol; further preferably, the organic solvent is anhydrous ethanol; in the premix, the mass concentration of the fluorinated polymer is 2-200 mg / mL; further preferably, in the premix, the mass concentration of the fluorinated polymer is 100 mg / mL; Preferably, the buffer is acetic acid-sodium acetate buffer or citric acid-sodium citrate buffer; the buffer pH is 4-8; further preferably, the buffer is acetic acid-sodium acetate buffer; the buffer pH is 4-6; most preferably, the buffer pH is 5.5; Preferably, the volume ratio of the premix to the buffer is 1:8 to 1:100; further preferably, the volume ratio of the premix to the buffer is 1:30; Preferably, the premixed solution is added dropwise into the buffer solution under vortex conditions to obtain a mixed solution, which is a polymer-lipid hybrid nanoparticle dispersion and can be used directly later; alternatively, the obtained mixed solution is dried to obtain polymer-lipid hybrid nanoparticles.

9. Use of the fluorinated polymer according to any one of claims 1 to 4 or the polymer-lipid hybrid nanoparticles according to any one of claims 6 to 7, characterized in that: For selective delivery of nucleic acid drugs to the spleen; Preferably, the fluorinated polymer or polymer-lipid hybrid nanoparticles are used for selective delivery of nucleic acid drugs to splenic dendritic cells; Preferably, the nucleic acid drug is selected from one or a combination of two or more of mRNA, small interfering RNA (siRNA), microRNA (microRNA), antisense oligonucleotide (ASO), circular RNA (circRNA), short hairpin RNA (shRNA), DNA or nucleic acid aptamers.

10. The use according to claim 9, characterized in that: The specific application methods are as follows: ① Adding the nucleic acid drug to the above polymer lipid hybrid nanoparticle dispersion, mixing thoroughly, and standing at room temperature to obtain a polymer-lipid hybrid nanoparticle-nucleic acid complex; ② The polymer-lipid hybrid nanoparticle-nucleic acid complex is administered to an organism to achieve spleen-selective delivery of nucleic acid drugs in the organism.

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