Fluorine-containing polymers, polymeric lipid hybrid nanoparticles for spleen-selective nucleic acid delivery, and methods of making and using the same

Abstract

The application provides a fluorine-containing polymer for spleen-selective nucleic acid delivery, a polymer-lipid hybrid nanoparticle and a preparation method and application thereof. The preparation method of the fluorine-containing polymer for spleen-selective nucleic acid delivery comprises the following steps: preparing the fluorine-containing polymer through ring-opening addition reaction of alkylene oxide, fluorine-substituted alkylene oxide and polyethylene imine. The preparation method of the fluorine-containing polymer is simple, the conditions are mild, and the yield of the finished product is high; the obtained fluorine-containing polymer is used for preparing the polymer-lipid hybrid nanoparticle, nucleic acid can be specifically delivered to the spleen and play a role, the polymer-lipid hybrid nanoparticle has a good transfection effect on spleen dendritic cells, and has good biological safety.

Description

Technical Field

[0001] This invention relates to a fluoropolymer, a polymer-lipid hybrid nanoparticle for selective nucleic acid delivery in the spleen, and its preparation method and application, belonging to the field of biomedical technology. Background Technology

[0002] In 2018, the FDA approved the first siRNA drug based on lipid / lipid-like nanoparticles (LNPs) (Onpattro); two years later, two SARS-CoV-2 vaccines based on mRNA-containing LNPs (Comirnaty, Spikevax) also entered clinics, saving millions of lives during the COVID-19 pandemic. Nucleic acid therapy is also receiving increasing attention from researchers.

[0003] RNA therapy encompasses the CRISPR-Cas9 system, mediated by antisense oligonucleotides (ASOs), small interfering RNA (siRNA), microRNAs (miRNAs), messenger RNA (mRNA), and single-lead RNAs (sgRNAs). These technologies can essentially manipulate any gene through various modes of action. However, RNA therapeutics are susceptible to nucleases and, due to their large size and negative charge, cannot penetrate cells. Current nucleic acid delivery vectors primarily include viral vectors, LNPs, polymer vectors, micelles, and vesicle vectors. LNPs or polymer vectors typically consist of four components: ionizable lipids (or amine-rich polymer structures), phospholipids, cholesterol, and polyethylene glycol-modified lipids. Ionizable lipids play a major role in protecting RNA and facilitating its cytoplasmic transport. Ionizable lipids are positively charged at acidic pH to condense RNA into LNPs, but neutral at physiological pH to reduce toxicity. After cellular uptake, they can be protonated in acidic endosomes and interact with anionic phospholipids in vivo, forming pyramidal ion pairs incompatible with the bilayer. These cationic and anionic lipids can drive the transition from a bilayer structure to the inverted hexagonal HII phase, thereby promoting membrane fusion / disruption, endosome escape, and cargo release into the cytoplasm.

[0004] Current delivery vectors for RNA delivery still have many shortcomings, including polar immune responses and long-term toxicity caused by ionizable lipids in liver nuclei (LNPs), and cumbersome synthesis processes. Meanwhile, 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. While there are reports of delivery vectors selectively delivering nucleic acids to the lungs, they cannot effectively selectively deliver nucleic acids to the spleen, which will severely limit nucleic acid therapy for spleen-related diseases.

[0005] Therefore, the rational design of ionizable lipids with targeting functions, which are easy 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] To overcome the shortcomings of existing technologies, this invention provides a fluoropolymer, polymer-lipid hybrid nanoparticles for selective nucleic acid delivery to the spleen, a preparation method thereof, and their applications. The fluoropolymer of this invention is prepared by ring-opening addition of polyethyleneimine, epoxides, and fluorinated epoxides; the preparation method is simple, the conditions are mild, and the yield is high; the obtained fluoropolymer is used to prepare polymer-lipid hybrid nanoparticles, which can specifically deliver nucleic acids to the spleen and exert their effects, exhibiting good transfection efficacy on splenic dendritic cells while also possessing good biocompatibility.

[0007] The technical solution adopted in this invention is as follows:

[0008] A method for preparing a fluoropolymer for spleen-selective nucleic acid delivery includes the steps of: preparing a fluoropolymer by ring-opening addition reaction of an epoxide, a fluorinated epoxide, and a polyethyleneimine.

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

[0010] According to a preferred embodiment of the present invention, the weight-average molecular weight (Mw) of polyethyleneimine (PEI) is 300-25000.

[0011] According to the present invention, preferably, the molar ratio of polyethyleneimine (PEI) to the total molar ratio of alkylene oxides and fluorinated alkylene oxides is 1:6-1:14; preferably, the molar ratio of polyethyleneimine (PEI) to the total molar ratio of alkylene oxides and fluorinated alkylene oxides is 1:9-1:13; and most preferably, the molar ratio of polyethyleneimine (PEI) to the total molar ratio of alkylene oxides and fluorinated alkylene oxides is 1:11.

[0012] According to the present invention, the molar ratio of epoxide to fluorinated epoxide is preferably 20:80-80:20; more preferably, the molar ratio of epoxide to fluorinated epoxide is 20:80-40:60; and most preferably, the molar ratio of epoxide to fluorinated epoxide is 40:60.

[0013] According to the present invention, preferably, the hydrocarbon group in the epoxide is selected from aliphatic hydrocarbon groups with saturated, unbranched, and unhalogenated carbon atoms having a number of C1 to C20; preferably, the hydrocarbon group in the epoxide is selected from aliphatic hydrocarbon groups with saturated, unbranched, and unhalogenated carbon atoms having a number of C4 to C18; more preferably, the epoxide is selected from one of 1,2-epoxidehexane, 1,2-epoxideoctane, 1,2-epoxidedecane, 1,2-epoxidedodecane, or 1,2-epoxidetetradecane.

[0014] According to the present invention, preferably, the hydrocarbon group in the fluorinated epoxide is selected from saturated, unbranched, or perfluorinated aliphatic hydrocarbon groups with a carbon number of C1-C20; preferably, the hydrocarbon group in the fluorinated epoxide is selected from saturated, unbranched, or perfluorinated aliphatic hydrocarbon groups with a carbon number of C4-C18; more preferably, the fluorinated epoxide is selected from 2,2,3,3,4,4,5,5,6,6,7,7,7-tridecylfluoroheptyl ethylene oxide (F9), 3-(perfluoron-octyl)-1,2-epoxypropane (F11), or 1H,2H,3H,3H-perfluoro-1,2-epoxytridecane (F13).

[0015] According to a preferred embodiment of the present invention, the ring-opening addition reaction temperature is 70-100°C and the ring-opening addition reaction time is 36-75 hours; more preferably, the ring-opening addition reaction temperature is 85-90°C and the ring-opening addition reaction time is 48-60 hours.

[0016] According to a preferred embodiment of the present invention, the ring-opening addition reaction is carried out in a light-protected environment under stirring conditions.

[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 fluoropolymer for spleen-selective nucleic acid delivery is prepared by the above method.

[0019] A polymer-lipid hybrid nanoparticle comprises the following raw material composition by weight percentage: 20-80% fluoropolymer, 2-40% PEGylated lipid, 5-60% steroid, and 0-10% auxiliary lipid.

[0020] According to a preferred embodiment of the present invention, the PEGylated lipid is selected from one of distearylphosphatidylethanolamine-PEG 2000, 1,2-dimyristoyl-rac-glycerol-3-methoxy-PEG 2000, d-α-succinate tocopheryl polyethylene glycol ester, 2-stannoyl-2-myristate-3-phosphatidylethanolamine-PEG 2000, dipalmitoylphosphatidylethanolamine-PEG 2000, or 1,2-distearyl-rac-glycerol-PEG 2000; preferably, the PEGylated lipid is one of distearylphosphatidylethanolamine-PEG 2000 or 1,2-dimyristoyl-rac-glycerol-3-methoxy-PEG 2000.

[0021] According to a preferred embodiment of the present invention, the steroid is selected from cholesterol, β-sitosterol, stigmasterol, stigmasterol, stigmasterol, ergosterol, cholesterol, dehydrocholesterol, dihydrocholesterol, hydroxycholesterol, or phytosterol; preferably, the steroid is selected from cholesterol, β-sitosterol, stigmasterol, stigmasterol, ergosterol, or phytosterol; most preferably, the steroid is cholesterol.

[0022] According to a preferred embodiment of the present invention, the auxiliary lipid is selected from one of 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC), 1,2-dioleoyl-SN-glycerol-3-phosphoethanolamine (DOPE), 1,2-distearyl-sn-propanetriyl-3-phosphocholine (DSPC), 1,2-dimyristoyl-sn-glycerol-3-phosphate-rac-(1-glycerol) (DPPG), 1,2-distearyl-sn-propanetriyl-3-phosphatidylethanolamine (DSPE), 1,2-dipalmitoyl-rac-glycerol-3-phosphocholine (DPPC), dimyristoylphosphatidylcholine (DMPC), or 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC); preferably, the auxiliary lipid is DOPE.

[0023] The preparation method of the above-mentioned polymer-lipid hybrid nanoparticles includes the following steps:

[0024] ① Dissolve the fluoropolymer, polyethylene glycol-modified lipids, steroids, and auxiliary lipids in an organic solvent to obtain a premix;

[0025] ② The premixed solution is added dropwise to the buffer solution under vortex conditions to obtain polymer lipid hybrid nanoparticles.

[0026] According to a preferred embodiment of the present invention, in step ①, the organic solvent is anhydrous ethanol or anhydrous methanol; preferably, the organic solvent is anhydrous ethanol; the mass concentration of the fluoropolymer in the premix is ​​2-200 mg / mL; preferably, the mass concentration of the fluoropolymer in the premix is ​​100 mg / mL.

[0027] According to the present invention, preferably, the buffer solution is an acetate-sodium acetate buffer solution or a citrate-sodium citrate buffer solution; the pH of the buffer solution is 4-8; preferably, the buffer solution is an acetate-sodium acetate buffer solution; the pH of the buffer solution is 4-6; most preferably, the pH of the buffer solution is 5.5.

[0028] According to the present invention, the volume ratio of the premixed solution to the buffer solution is preferably 1:8 to 1:100; more preferably, the volume ratio of the premixed solution to the buffer solution is 1:30.

[0029] According to a preferred embodiment of the present invention, the mixture obtained by adding the premixed solution dropwise to the buffer solution under vortex conditions is a polymer lipid hybrid nanoparticle dispersion, which can be used directly thereafter; or, the obtained mixture is dried to obtain polymer lipid hybrid nanoparticles.

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

[0031] According to a preferred embodiment of the present invention, the fluoropolymer or polymer-lipid hybrid nanoparticles are used for selective delivery of nucleic acid drugs to splenic dendritic cells.

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

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

[0034] ① Add the nucleic acid drug to the above polymer-lipid hybrid nanoparticle dispersion, mix thoroughly, and let stand at room temperature to obtain a polymer-lipid hybrid nanoparticle-nucleic acid complex;

[0035] ② The above polymer-lipid hybrid nanoparticle-nucleic acid complex is applied 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 nanoparticles to the nucleic acid drug is 2:1-30:1; more preferably, the mass ratio of the polymer lipid hybrid nanoparticles to the nucleic acid drug is 5:1-20:1; further preferably, the mass ratio of the polymer lipid hybrid nanoparticles to the nucleic acid drug is 10:1-20:1; most preferably, the mass ratio of the polymer lipid hybrid nanoparticles to the nucleic acid drug is 15:1.

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

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

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

[0040] (1) The raw materials of this invention are readily available, and the preparation process is mild, simple, safe, and yields a high product yield.

[0041] (2) This invention introduces fluorinated alkylene oxide segments into polymers of alkylene oxides and polyethyleneimine, enabling the polymer lipid hybrid nanoparticles prepared by this invention to specifically deliver nucleic acid drugs to spleen dendritic cells and exert their effects, exhibiting excellent transfection efficacy and good biocompatibility. The addition of fluorinated alkylene oxides can enhance the uptake of nanoparticles by cells in vivo and in vitro, and enhance endosome escape, thereby enhancing 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 protein crowns 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 the need to add active targeting molecules or auxiliary components, thus effectively reducing the application cost.

[0043] (4) The targeted delivery of splenic dendritic cells in this invention provides a possibility and guidance for realizing the immunotherapy of 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 fluorinated alkyl oxyoxides are used instead of alkyl oxyoxides, the resulting nanoparticles have poor dispersibility and cannot achieve nucleic acid drug delivery. If the molar ratio of alkyl oxyoxides to fluorinated alkyl oxyoxides is not suitable, the selective delivery and transfection effect of the nanoparticles will be reduced. Similarly, if the type of fluorinated alkyl oxyoxide or the type of alkyl oxyoxide is unsuitable, the selective delivery and transfection effect of the nanoparticles will also be reduced. Attached Figure Description

[0046] Figure 1 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 Encapsulation efficiency of polymer-lipid hybrid nanoparticles for mRNA (a) and their cytotoxicity to HeLa cells (b).

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

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

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

[0051] Figure 6 Flow cytometry pseudocolor density map of spleen cell subset mRNA delivery mediated by polymer-lipid hybrid nanoparticles (a) and quantitative analysis of the proportion of DiO positive cells in major spleen cell subsets (b).

[0052] Figure 7 Histopathological reports of the heart, liver, spleen, lungs, and kidneys 24 hours after intravenous injection of polymer-lipid hybrid nanoparticles loaded with mRNA into mice. Detailed Implementation

[0053] The technical solution of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods used in the embodiments are conventional methods. Unless otherwise specified, the materials, reagents, etc. used in the embodiments can be obtained commercially.

[0054] In the examples, the polyethyleneimine is branched polyethyleneimine with a weight-average molecular weight (Mw) of 600.

[0055] Example 1

[0056] A method for preparing a fluoropolymer for spleen-selective nucleic acid delivery includes the following steps:

[0057] 1,2-Epoxydecane and 2,2,3,3,4,4,5,5,6,6,7,7,7-Tetrafluoroheptylethylene oxide were mixed uniformly at a molar ratio of 80:20 to obtain an epoxy alkane premix. Polyethyleneimine was added and mixed uniformly, wherein the molar ratio of polyethyleneimine to the total molar ratio of 1,2-epoxydecane and tridecafluoroheptylethylene oxide was 1:11. The mixture was stirred continuously at 90°C for 60 hours under light-protected conditions. The resulting crude product was dialyzed against anhydrous ethanol and then dried at 50°C to remove the ethanol, yielding a fluoropolymer.

[0058] Example 2

[0059] A method for preparing a fluoropolymer for spleen-selective nucleic acid delivery includes the following steps:

[0060] 1,2-Epoxydecane and 2,2,3,3,4,4,5,5,6,6,7,7,7-Tetrafluoroheptylethylene oxide were mixed uniformly at a molar ratio of 40:60 to obtain an epoxy alkane premix. Polyethyleneimine was added and mixed uniformly, wherein the molar ratio of polyethyleneimine to the total molar ratio of 1,2-epoxydecane and tridecafluoroheptylethylene oxide was 1:11. The mixture was stirred continuously at 90°C for 60 hours under light-protected conditions. The resulting crude product was dialyzed against anhydrous ethanol and then dried at 50°C to remove the ethanol, yielding a fluoropolymer.

[0061] Example 3

[0062] A method for preparing a fluoropolymer for spleen-selective nucleic acid delivery includes the following steps:

[0063] 1,2-Epoxydecane and 2,2,3,3,4,4,5,5,6,6,7,7,7-Tetrafluoroheptylethylene oxide were mixed uniformly at a molar ratio of 20:80 to obtain an epoxy alkane premix. Polyethyleneimine was added and mixed uniformly, wherein the molar ratio of polyethyleneimine to the total molar ratio of 1,2-epoxydecane and tridecafluoroheptylethylene oxide was 1:11. The mixture was stirred continuously at 90°C for 60 hours under light-protected conditions. The resulting crude product was dialyzed against anhydrous ethanol and then dried at 50°C to remove the ethanol, yielding a fluoropolymer.

[0064] Example 4

[0065] A method for preparing a fluoropolymer for spleen-selective nucleic acid delivery, as described in Example 2, except that 2,2,3,3,4,4,5,5,6,6,7,7,7-tridecylfluoroheptyl ethylene oxide is replaced with 3-(perfluorooctyl)-1,2-epoxypropane; other steps and conditions are the same as in Example 2.

[0066] Comparative Example 1

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

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

[0069] Comparative Example 2

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

[0071] Polyethyleneimine was mixed with 2,2,3,3,4,4,5,5,6,6,7,7,7-tridecylfluoroheptylethylene oxide at a molar ratio of 1:11 and stirred continuously at 90°C for 60 hours under light-protected conditions. The crude product was dialyzed against anhydrous ethanol and then dried at 50°C to remove the ethanol, yielding the polymer.

[0072] Example 5

[0073] A polymer lipid hybrid nanoparticle comprises the following raw material composition in weight percentages: 57% fluoropolymer (prepared in Example 1), 28.5% polyethylene glycol-modified lipid (1,2-dimyristic-rac-glycerol-3-methoxy-polyethylene glycol 2000, DMG-PEG), and 14.5% steroid (cholesterol).

[0074] The preparation method of the above polymer-lipid hybrid nanoparticles includes the following steps:

[0075] Fluoropolymer, DMG-PEG, and cholesterol were dissolved in anhydrous ethanol to obtain a lipid premix (the fluoropolymer concentration was 100 mg / mL). This lipid premix was then added dropwise to an acetate-sodium acetate buffer solution at pH 5.5 (the volume ratio of the lipid premix to the buffer solution was 1:30) under vortex conditions 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 fluoropolymer prepared in Example 1 is replaced with the fluoropolymer prepared in Example 2; the other raw material composition is the same as in Example 5.

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

[0079] Example 7

[0080] A polymer lipid hybrid nanoparticle, as described in Example 5, except that the fluoropolymer prepared in Example 1 is replaced with the fluoropolymer prepared in Example 3; the other raw material composition is the same as in Example 5.

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

[0082] Example 8

[0083] A polymer lipid hybrid nanoparticle, as described in Example 5, except that the fluoropolymer prepared in Example 1 is replaced with the fluoropolymer prepared in Example 4; the other raw material composition is the same as in Example 5.

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

[0085] Comparative Example 3

[0086] A polymer lipid hybrid nanoparticle, as described in Example 5, except that the fluorinated polymer prepared in Example 1 is replaced with the polymer prepared in Comparative Example 1; the other raw material composition is the same as in Example 5.

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

[0088] Comparative Example 4

[0089] A polymer lipid hybrid nanoparticle, as described in Example 5, except that the fluorinated polymer prepared in Example 1 is replaced with the fluorinated polymer prepared in Comparative Example 2; the other raw material composition is the same as in Example 5.

[0090] The preparation method of the above-mentioned polymer-lipid hybrid nanoparticles is the same as described in Example 5, except that the polymer-lipid hybrid nanoparticle dispersion was 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 rapidly and uniformly mixed with mRNA, wherein the mass ratio of polymer-lipid hybrid nanoparticles to mRNA was 5:1, 10:1, 15:1, and 20:1, respectively; after being incubated at room temperature for 20 minutes, polymer-lipid hybrid nanoparticle-nucleic acid complexes were obtained. The sample with a polymer-lipid hybrid nanoparticle to mRNA mass ratio of 15:1 was named PEI-F20 / mRNA.

[0094] Application Example 2

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

[0096] Application Example 3

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

[0098] Application Example 4

[0099] An application of polymer-lipid hybrid nanoparticles is described in Application Example 1, except that the polymer-lipid hybrid nanoparticle dispersion prepared in Example 5 is replaced with the polymer-lipid hybrid nanoparticle dispersion prepared in Example 8; 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 described in Application Example 1, except that the polymer-lipid hybrid nanoparticle dispersion prepared in Example 5 is replaced with the polymer-lipid hybrid nanoparticle dispersion prepared in Comparative Example 3; other steps and conditions are the same as in Application Example 1. The sample with a polymer-lipid hybrid nanoparticle to mRNA mass ratio of 15:1 is named PEI-H / mRNA.

[0102] Application Comparative Example 6

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

[0104] Experimental Example 1

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

[0106] Experimental Example 2

[0107] The encapsulation efficiency of nanoparticles on mRNA in polymer-lipid hybrid nanoparticle-nucleic acid complexes prepared in corresponding use cases 1-3 and application comparison example 5 was quantitatively analyzed.

[0108] like Figure 2 As shown in (a), PEI-H, PEI-F20, and PEI-F60 all achieved an encapsulation rate of over 80% for mRNA, while the encapsulation rate of PEI-F80 dropped sharply to around 50% when the mass ratio was 5:1.

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

[0110] Experimental Example 3

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

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

[0113] Test Example 4

[0114] The polymer-lipid hybrid nanoparticle-nucleic acid complexes (where mRNA was replaced with mLuc, and the mass ratio of polymer-lipid hybrid nanoparticles to mLuc was 15:1) prepared in Application Example 2 and Comparative Example 5 were intravenously injected into mice at a dose of 0.2 μg / g of mLuc. The expression of mLuc in mice was observed using a small animal imaging system 6 hours after intravenous injection.

[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 various organs, including the heart, liver, spleen, lungs, and kidneys. PEI-F60 showed good mLuc expression in the mouse spleen.

[0116] Experimental Example 5

[0117] The polymer-lipid hybrid nanoparticle-nucleic acid complexes (with a polymer-lipid hybrid nanoparticle to mRNA mass ratio of 15:1) prepared in Application Example 2 and Comparative Example 5 were intravenously injected into mice at a dose of 0.2 μg / g mRNA. Six hours after intravenous injection, the cellular selectivity of PEI-H and PEI-F60 for spleen-targeted mRNA delivery was examined. Results are as follows... Figure 6 As shown, DIO-labeled PEI-H and PEI-F60 were used to deliver mouse spleen cell populations. Different types of spleen cells were identified by immunophenotyping using corresponding markers or antigens, including macrophages (CD45+), epithelial cells (CD326+), endothelial cells (CD31+), and dendritic cells (CD11c+). Fluorescent activated cell sorting analysis revealed that approximately 30% of the dendritic cells delivered by PEI-F60 could be labeled, demonstrating potential for immunotherapy.

[0118] The polymer-lipid hybrid nanoparticle-nucleic acid complexes (where the mass ratio of polymer-lipid hybrid nanoparticles to mRNA was 15:1) prepared in Application Example 2 and Comparative Example 5 were intravenously injected into mice at a dose of 0.2 μg / g mRNA. Twenty-four hours later, the heart, liver, spleen, lungs, and kidneys of the mice were subjected to H&E staining for pathological examination. Figure 7 As shown, compared with untreated mice, no obvious lesions were observed in any organ of the mice treated with the complex, indicating that the polymer-lipid hybrid nanoparticles of the present invention have good biosafety in vivo at the dose required to achieve effective transfection.

Claims

1. An application of polymer lipid hybrid nanoparticles, characterized in that, Used for selective delivery of nucleic acid drugs to the spleen; The polymer lipid hybrid nanoparticles are characterized by comprising the following raw material composition by mass percentage: 20-80% fluoropolymer, 2-40% polyethylene glycol-modified lipid, 5-60% steroid, and 0-10% auxiliary lipid. The method for preparing the fluoropolymer includes the following steps: fluoropolymer is prepared by ring-opening addition reaction of alkyl epoxide, fluorinated alkyl epoxide, and polyethyleneimine; the molar ratio of alkyl epoxide to fluorinated alkyl epoxide is 20:80-80:20; the fluorinated alkyl epoxide is selected from 2,2,3,3,4,4,5,5,6,6,7,7,7-tridecylfluoroheptylethylene oxide (F9).

2. The application according to claim 1, characterized in that, Includes one or more of the following conditions: i. Polyethyleneimine (PEI) is either branched polyethyleneimine (BPEI) or linear polyethyleneimine (LPEI); ii. Polyethyleneimine (PEI) has a weight-average molecular weight (Mw) of 300-25000; iii. The molar ratio of polyethyleneimine (PEI) to the total molar ratio of epoxides and fluorinated epoxides is 1:6 to 1:

14. iv. The hydrocarbon group in epoxides is selected from saturated, unbranched, and unhalogenated aliphatic hydrocarbon groups with C1 to C20 carbon atoms; v. The ring-opening addition reaction temperature is 70-100℃, and the ring-opening addition reaction time is 36-75 hours; vi. The ring-opening addition reaction is carried out in a dark environment under stirring conditions.

3. The application according to claim 2, characterized in that, Includes one or more of the following conditions: i. The molar ratio of polyethyleneimine (PEI) to the total molar ratio of epoxides and fluorinated epoxides is 1:9-1:

13. ii. The hydrocarbon groups in epoxides are selected from saturated, unbranched, and unhalogenated aliphatic hydrocarbon groups with C4 to C18 carbon atoms; iii. The ring-opening addition reaction temperature is 85-90℃, and the ring-opening addition reaction time is 48-60 hours.

4. The application according to claim 1, characterized in that, The molar ratio of epoxides to fluorinated epoxides is 20:80-40:

60.

5. The application according to claim 4, characterized in that, The molar ratio of epoxides to fluorinated epoxides is 40:

60.

6. The application according to claim 1, characterized in that, Includes one or more of the following conditions: i. The PEGylated lipid is selected from one of the following: distearylphosphatidylethanolamine-PEG 2000, 1,2-dimyristoyl-rac-glycerol-3-methoxy-PEG 2000, d-α-succinate tocopherol PEG ester, 2-stannyl-myristate-3-phosphatidylethanolamine-PEG 2000, dipalmitoylphosphatidylethanolamine-PEG 2000, or 1,2-distearyl-rac-glycerol-PEG 2000; ii. The steroid is selected from one of cholesterol, β-sitosterol, stigmasterol, stigmasterol, stigmasterol, ergosterol, cholesterol, dehydrocholesterol, dihydrocholesterol, hydroxycholesterol, or lypotassium. iii. The auxiliary lipid is selected from one of 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), 1,2-dioleoyl-SN-glycerol-3-phosphoethanolamine (DOPE), 1,2-distearyl-sn-propanetriyl-3-phosphate choline (DSPC), 1,2-dimyristoyl-sn-glycerol-3-phosphate-rac-(1-glycerol) (DPPG), 1,2-distearyl-sn-propanetriyl-3-phosphatidylethanolamine (DSPE), 1,2-dipalmitoyl-rac-glycerol-3-phosphate choline (DPPC), dimyristoylphosphatidylcholine (DMPC), or 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (POPC).

7. The application according to claim 1, characterized in that, The preparation method of polymer lipid hybrid nanoparticles includes the following steps: ① Dissolve the fluoropolymer, polyethylene glycol-modified lipids, steroids, and auxiliary lipids in an organic solvent to obtain a premix; ② The premixed solution is added dropwise to the buffer solution under vortex conditions to obtain polymer lipid hybrid nanoparticles.

8. The application according to claim 7, characterized in that, In step ①, the organic solvent is anhydrous ethanol or anhydrous methanol; the mass concentration of the fluoropolymer in the premix is ​​2-200 mg / mL; the buffer is acetate-sodium acetate buffer or citrate-sodium citrate buffer; the pH of the buffer is 4-8; the volume ratio of the premix to the buffer is 1:8 to 1:100; the mixture obtained by adding the premix dropwise to the buffer under vortex conditions is the polymer lipid hybrid nanoparticle dispersion, which can be used directly thereafter; or, the obtained mixture can be dried to obtain polymer lipid hybrid nanoparticles.

9. The application according to claim 1, characterized in that, The fluoropolymer or polymer-lipid hybrid nanoparticles are used for selective delivery of nucleic acid drugs to splenic dendritic cells; the nucleic acid drug is selected from one or more combinations of spontaneous RNA (mRNA), small interfering RNA (siRNA), microRNA, antisense oligonucleotide (ASO), circular RNA (circRNA), short hairpin RNA (shRNA), DNA, or nucleic acid aptamers.

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

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