Preparation method of polymer-lipid composition capable of mediating efficient transfection of nucleic acid and product thereof

Abstract

The invention provides a polymer-lipid composition capable of mediating efficient transfection of nucleic acid and a preparation method thereof, and in some embodiments, the invention provides a safe and stable polymer-lipid composition capable of mediating efficient transfection of nucleic acid in vivo after being administered through a respiratory route. And the nucleic acid transfection is particularly useful in improving the nucleic acid transfection in bronchial epithelial cells and lung dendritic DC cells.

Description

Technical Field

[0001] The invention relates to the field of pharmaceutical preparations, in particular to a method for preparing a polymer-lipid composition capable of mediating efficient nucleic acid transfection, and also to a product prepared by the method. Background Art

[0002] In recent years, messenger RNA (mRNA) has shown great potential as a new therapeutic tool in the treatment of various diseases, especially in the rapid response to emerging pathogens. In vitro transcribed mRNA technology has revolutionized the biomedical field due to its record-breaking development speed and its flexible design to express any target protein. The currently approved COVID-19 mRNA vaccines have been administered billions of times, saved millions of lives, won family recognition, and became the subject of the 2023 Nobel Prize.

[0003] Lipid nanoparticles (LNP) are the most advanced mRNA therapeutic delivery system in the clinic, and their effectiveness has been confirmed in multiple clinical cases. With many advantages, LNP has developed into the "gold standard" delivery technology in the current mRNA field. However, although the traditional LNP system plays a certain role in protecting mRNA from rapid degradation in the body, it tends to accumulate in the liver and spleen after intramuscular or intravenous injection, which greatly limits the widespread application of mRNA therapy in other target organ disease fields. There are many respiratory diseases, such as pandemics caused by respiratory pathogens such as influenza virus and coronavirus, asthma, cystic fibrosis (CF), etc. The main lesions are in the trachea, bronchi, lungs and chest cavity, which is one of the most likely targets of mRNA therapy. However, the microenvironment of respiratory mucosal tissue is complex and has a physiological barrier to efficiently remove exogenous foreign bodies, which makes it difficult for LNP to mediate efficient transfection of mRNA in the respiratory tract. Therefore, there is an urgent need to develop a new delivery system for efficient delivery of mRNA molecules. Summary of the invention

[0004] The first object of the present invention is that polymer-lipid compositions can mediate mRNA delivery more efficiently than lipid nanoparticles (LNPs).

[0005] The second purpose of the present invention is to significantly reduce the toxic effects of lipid nanocarriers after adding polymers.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] The invention provides a polymer-lipid composition capable of mediating efficient mRNA transfection, wherein the composition comprises messenger RNA (mRNA), a polymer, a cationic lipid and a non-cationic lipid.

[0008] Preferably, the polymer comprises at least one of an amphiphilic polymer, a hydrophilic polymer or a hydrophobic polymer; the polymer accounts for 2%-90% by weight of the composition, more preferably 15%-80% by weight, more preferably 35%-70% by weight, more preferably 45%-70% by weight, more preferably 45%-60% by weight, and most preferably 50%-60% by weight. Specifically, it can be 10%, 17.8%, 41.4%, 52%, 54.1%, 68.4%, 70.2%, 81.2%.

[0009] Preferably, the amphiphilic polymer is selected from one or more of O,O′-bis(2-aminopropyl)polypropylene glycol-block-polyethylene glycol-block-polypropylene glycol, polylactic acid-glycolic acid copolymer, polyethylene glycol-b-polycaprolactone containing or not containing functional groups, polylactic acid-polyethylene glycol containing or not containing functional groups, polyarylether-polycaprolactone containing or not containing functional groups, polyethylene glycol-b-polycaprolactone-b-polyamino ester, polyethylene glycol-polylactic acid-glycolic acid copolymer, lauryl alcohol polyether, and methacrylate copolymer.

[0010] Preferably, the hydrophilic polymer is selected from one or more of polyethylene glycol, four-arm polyethylene glycol containing or not containing functional groups, eight-arm polyethylene glycol containing or not containing functional groups, polyvinyl alcohol, polyacrylamide, branched polyethyleneimine, linear polyethyleneimine, glycerol and derivatives, dipropylene glycol, gelatin, hydroxypropyl methylcellulose, carbomer, chitosan and derivatives thereof.

[0011] Preferably, the hydrophobic polymer is selected from one or more of polylactic acid, polypropylene glycol, polyetheramine, polycaprolactone, polyvinyl pyrrolidone, cellulose acetate phthalate, and acrylic resin.

[0012] Preferably, the nucleic acid comprises one or more selected from the group consisting of mRNA, self-amplifying RNA (saRNA), circular RNA (circRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA) and micro RNA (miRNA), primary-miRNA, antisense oligonucleotide (ASO), transfer RNA (tRNA), plasmid DNA (pDNA), single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), deoxyribozyme (DNAzyme), ribozyme (RNAzyme), nucleic acid aptamer, clustered regularly interspaced short palindromic repeats (CRISPR)-related nucleic acid, single guide RNA (sgRNA), CRISPR-RNA (crRNA), trans-activating crRNA (tracrRNA), guide RNA, single-stranded RNA (ssRNA) and double-stranded RNA (dsRNA).

[0013] Preferably, the cationic lipid is selected from one or more of permanent cationic lipids and ionizable cationic lipids; the molar ratio of the cationic lipid to the total lipid is 35%-65%, more preferably 40%-60%, more preferably 45%-55%, or most preferably 45%-50%.

[0014] Preferably, the cationic lipid is selected from DOTMA, DOSPA, DOTAP, ePC, DODAP, DODMA, DDAB, DSDMA, DODAC, DOAP, DMRIE, DOGS, DMOBA, HGT5000, HGT5001, HGT5002, HGT4001, HGT4002, HGT4003, HGT4005, DLin-MC3-DMA, DLin-KC2-DMA, Acuitas ALC-0315, AcuitasA9, Acuitas Lipid 2,2, Moderna Lipid H (SM-102), Moderna Lipid 5. A2-Iso5-2DC18, BAME-O16B, 9A1P9, C12-200, cKK-E12, OF-Deg-Lin, 306Oi10, TT3, FTT5, Lipid319, 5A2-SC8, Genevant CL1, DLinDMA, DLenDMA, ClinDMA, CpLinDMA, imidazole cholesteryl ester (ICE), RE-1, RE-2, RE-3, GL-67, 5A2-SC8, Acuitas A9, Arcturus Lipid 2,2(8,8)4C CH3, OF-02, A18-Iso5-2DC18, BAME-O16B, A6, 98N12-5, L319, L343, 304O13, 306O138, 306O12B, 30 6-O12B, LP01, G0-C14, 7C1, Cephalin, Dlin-EG-DMA, DLinAP, DLin-MPZ, DLin-C-DAP, DLin-2-DMAP , Dlin-S-DMA, DLinDAP, DLin-MA, DLin-DAC, DLin-K-DMA, DLin-K-MPZ, DLin-K-DMA, DLin-K6-C4-DMA, DLin-K-C4-DMA, DLin-K-C3-DMA, CpLinDMA, DOcarbDAP, DLincarbDAP, C12-(2-3-2), Genevant Lipid One or more of CL1, XTC, ALNY-100, NC98-5 and their derivatives.

[0015] Preferably, the non-cationic lipid is selected from one or more of neutral lipids, PEG lipids and structured lipids.

[0016] Preferably, the neutral lipid is selected from lecithin, phosphatidylethanolamine, lysophosphatidylcholine, lysophosphatidylethanolamine, phosphatidylserine, dioleoylphosphatidylserine (DOPS), phosphatidylinositol, sphingomyelin, egg yolk sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dihexadecyl phosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylethanolamine (DOPE), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleoyl-phosphatidylglycerol (POPG), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-methyl ester (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, dioleoyl-phosphatidylethanolamine (DEPE), stearoyloleoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine, egg yolk phosphatidylcholine (EPC), dilinoleoylphosphatidylcholine, 1,2-dipalmitoyl-sn-glycero-3-O-4'-(N,N,N-trimethyl)-homoserine (DGTS), monogalactosyldiacylglycerol (MGDG), diacetyldiacylglycerol (DGDG), sulfonylquinolinediacylglycerol (SQDG), 1-palmitoyl-2-cis-9,10-methylenehexyl-decanoyl-sn-glycero-3-phosphocholine (Cyclo PC), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, MSPC, DMPC, DLPC, DPPS, DUPC and one or more of their derivatives; the molar ratio of the neutral lipids to the total lipids is 5%-30%, the more preferred molar ratio is 10%-25%, the more preferred molar ratio is 10%-20%, and the most preferred molar ratio is 15%-20%.

[0017] Preferably, the PEG lipid is selected from one or more of PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DSPE, ceramide-PEG2000, ALC-0159, DSPE-PEG, and PEG-PE; the molar ratio of the PEG lipid to the total lipid is 0%-5%, more preferably 0%-3%, more preferably 0%-1.5%, more preferably 0%-1%, more preferably 0.3%-1%, and more preferably 0.5%-1%.

[0018] Preferably, the structural lipids are selected from one or more of cholesterol, coprosterol, sitosterol, ergosterol, campesterol, fucoxanthin, β-sitosterol, stigmasterol, brassicasterol, tomatine, tomatine, ursolic acid, and α-tocopherol; the molar ratio of the structural lipids to the total lipids is 20%-45%, a more preferred molar ratio is 25%-40%, a more preferred molar ratio is 25%-35%, and the most preferred molar ratio is 25%-30%.

[0019] Preferably, the polymer-lipid composition for delivering mRNA is administered via the respiratory tract, or orally, or intramuscularly, or intravenously, or subcutaneously, or intratumorally.

[0020] Preferably, the method for preparing the composition comprises the following steps: adding an mRNA solution or a lipid solution according to the water solubility and lipid solubility characteristics of the polymer, and then mixing the mRNA solution (aqueous phase) and the lipid solution (organic phase); or first mixing the mRNA solution (aqueous phase) and the lipid solution (organic phase), purifying to obtain lipid nanoparticles LNP that stably encapsulate mRNA, and then mixing the LNP with the polymer.

[0021] In the present invention, the weight percentage of the polymer in the composition is 2%-90%; the molar ratio of the cationic lipid to the total lipid is 35%-65%; the molar ratio of the neutral lipid to the total lipid is 5%-30%; the molar ratio of the structural lipid to the total lipid is 20%-45%; and the molar ratio of the PEG lipid to the total lipid is 0%-3%.

[0022] In some of the embodiments, suitable polymers have an average molecular weight of about 200 g / mol-90,000 g / mol; in some of the embodiments, suitable polymers have an average molecular weight of about 600 g / mol-30,000 g / mol; in some of the embodiments, suitable polymers have an average molecular weight of about 8,000 g / mol-30,000 g / mol; in some of the embodiments, suitable polymers have an average molecular weight of about 1,000 g / mol-20,000 g / mol.

[0023] In some embodiments, the method for preparing polymer-lipid nanoparticles includes the step of adding the polymer to the mRNA solution (aqueous phase).

[0024] In some embodiments, the method for preparing polymer-lipid nanoparticles comprises the step of adding a polymer to a lipid solution (organic phase).

[0025] In some embodiments, the method for preparing polymer-lipid nanoparticles includes the step of adding a polymer to LNP.

[0026] In some embodiments, the molar ratio of the cationic lipid, neutral lipid, structural lipid and PEG lipid is 50:10:38.5:1.5, 49:20.1:30:0.9, 46:23:29.5:1.5, 60:20:29:1, 40:20:38:1, 54:20:25:1, 60:5:33:1, 45:10:44:1, 50:15:33.5:1.5, 40:32:27:1, 40:32:28:0, 49:20.7:30:0.3, 49:20.5:30:0.5, 49:19.5:30:1.5, 49:19:30:2, and 49:17:30:4.

[0027] In some of these embodiments, the polymer-lipid nanoparticles have an mRNA encapsulation efficiency of 85%-99%.

[0028] In some of these embodiments, the polymer-lipid nanoparticles have an average size of less than 150 nm and a polydispersity index (PDI) of less than 0.15.

[0029] In this application, the use of "or" means "and / or" unless otherwise stated. As used in this disclosure, the term "including" and variations of the term, such as "comprising" and "comprises", are not intended to exclude other additives, components, integers or steps. As used in this patent application, the terms "about" and "approximately" are used as equivalents. Both terms are intended to cover any normal fluctuations understood by ordinary technicians in the relevant fields.

[0030] Beneficial effects of the present invention: The present invention provides a polymer-lipid composition delivery system that can mediate efficient mRNA transfection. The delivery system has the advantages of simple preparation method, low cost, high cellular uptake, safety, ability to enhance mRNA lung delivery and ability to withstand shear stress during atomization, and can be used for the treatment of lung diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to make the purpose, technical solution and beneficial effects of the present invention clearer, the present invention provides the following drawings for illustration:

[0032] Figure 1 Particle size, PDI and encapsulation efficiency analysis of polymer-lipid delivery systems prepared based on hydrophilic polymers (A: particle size; B: PDI; C: encapsulation efficiency);

[0033] Figure 2Particle size, PDI and encapsulation efficiency analysis of polymer-lipid delivery systems prepared based on hydrophobic polymers (A: particle size; B: PDI; C: encapsulation efficiency);

[0034] Figure 3 Particle size, PDI and encapsulation efficiency analysis of polymer-lipid delivery systems prepared based on amphiphilic polymers (A: particle size; B: PDI; C: encapsulation efficiency);

[0035] Figure 4 Analysis of the anti-atomization shear ability of different types of polymer-lipid delivery systems (A: particle size; B: PDI; C: encapsulation efficiency);

[0036] Figure 5 Cellular uptake analysis for different types of polymer-lipid delivery systems;

[0037] Figure 6 To analyze the effectiveness of EGFP mRNA transfection mediated by different types of polymer-lipid delivery systems in cell models;

[0038] Figure 7 To analyze the effectiveness of MetLuc mRNA transfection mediated by different types of polymer-lipid delivery systems in cell models (A: DC 2.4 cells; B: A549 cells; C: 16HBE cells);

[0039] Figure 8 To analyze the cytotoxicity of different types of polymer-lipid delivery systems;

[0040] Fig. 9 The transfection effect of the polymer-lipid delivery system configured with different polymer concentrations (A: the transfection effect of the polymer-lipid delivery system configured with different concentrations of 4armPEG10K; B: the transfection effect of the polymer-lipid delivery system configured with different concentrations of PPG3K; C: the transfection effect of the polymer-lipid delivery system configured with different concentrations of PEG5K-b-PCL2K);

[0041] Fig.10 transfection effects of polymer-lipid delivery systems configured for different types of polymers;

[0042] Fig.11 The transfection effect of fLucmRNA encapsulated by physical mixed preparation;

[0043] Fig.12The transfection effects of polymer-lipid delivery systems configured with different N / P ratios and lipid ratios (A: transfection effect of PPG3K-lipid delivery systems configured with different N / P ratios, wherein SM-102, DSPC, Cholesterol and DMG-PEG2000 are in a molar ratio of 50:10:38.5:1.5; B: transfection effect of PPG3K-lipid delivery systems configured with different cationic lipid ratios, N / P=8; C: transfection effect of PPG3K-lipid delivery systems configured with different phospholipid ratios, N / P=8; D: transfection effect of PPG3K-lipid delivery systems configured with different PEG lipid ratios, N / P=8);

[0044] Fig.13 Transfection effects of PPG3K-lipid delivery systems configured with different types of lipids (A: transfection effects of PPG3K-lipid delivery systems configured with different cationic lipids; B: transfection effects of PPG3K-lipid delivery systems configured with different phospholipid lipids; C: transfection effects of PPG3K-lipid delivery systems configured with different PEG lipids; D: transfection effects of PPG3K-lipid delivery systems configured with different cholesterol);

[0045] Fig.14 Analysis of tissue targeting and cell targeting of polymer-lipid delivery system (PPG3K) (A: Representative results of the distribution of DID-labeled PPG3K-lipid delivery system in isolated organs 4 hours after intranasal administration (in) (left) and fluorescence quantitative results in lungs; B: lung epithelial cells (ECs), type I alveolar cells (T1PCs), neutrophils (NTs), macrophages in the above lungs and the proportion of DID fluorescence-positive cells in DCs);

[0046] Fig.15 Figure 3 is an analysis of the effectiveness of polymer-lipid delivery system in transfecting mRNA in tissues and cells (A: exemplary results of transfection mediated by PPG3K-lipid delivery system loaded with fLuc mRNA in living mice and isolated organs 6 hours after intranasal administration (in); B: quantitative results of Fluc bioluminescent signal in the above living mice and lungs; C: quantitative results of PPG3K-lipid delivery system loaded with EGFP mRNA in lung epithelial cells (ECs), type I alveolar cells (T1PCs), neutrophils (NTs), macrophages 6 hours after intranasal administration (in) and the proportion of green fluorescence-positive cells in DCs);

[0047] Fig.16Pharmacokinetic study of proteins expressed by mRNA mediated by polymer-lipid delivery system: (kinetic curve of luciferase expression in the lungs over time after intranasal administration (in) of PPG3K-lipid delivery system loaded with fLuc mRNA);

[0048] Fig.17 Safety investigation of polymer-lipid delivery system (A: Representative H&E staining results of mouse lung tissue sections 48 hours after non-nasal administration (in) of PPG3K-lipid delivery system, control samples of PBS solution and LNP preparation inoculated by in route were used as negative control and positive control respectively; B: 48 hours after inoculation of PPG3K-lipid delivery system by in route, serum was collected to detect the content of various biochemical indicators in mice);

[0049] Fig.18 Study on the immune effect of KPmRNA vaccine encapsulated by polymer-lipid delivery system (A: KP antigen-specific IgG antibody level detected in mouse serum samples 28 days after primary immunization (expressed as OD450 value); B: KP antigen-specific IgA antibody level detected in mouse alveolar lavage fluid samples 28 days after primary immunization (expressed as OD450 value); C: KP antigen-specific IgA antibody level detected in mouse nasal lavage fluid samples 28 days after primary immunization (expressed as OD450 value); D: Lung bacterial load of mice immunized with PBS, LNP and PPG3K-lipid preparations 2 days after infection with K1 type Klebsiella pneumoniae; E: Body weight changes of mice immunized with PBS, LNP and PPG3K-lipid preparations after infection with K1 type Klebsiella pneumoniae; F: Survival rate of mice immunized with PBS, LNP and PPG3K-lipid preparations after infection with K1 type Klebsiella pneumoniae). DETAILED DESCRIPTION

[0050] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0051] Example 1. Preparation of mRNA delivery system

[0052] The preparation method of the delivery system for stably encapsulating mRNA, the specific steps are as follows:

[0053] (a) Preparation of lipid nanoparticles LNP:

[0054] Preparation of organic phase: Weigh lipids using a 100,000th balance, dissolve lipids SM102, DSPC, Cholesterol and DMG-PEG2000 in ethanol at a molar ratio of 49:20.1:30:0.9 and mix, and dilute the total lipid concentration with ethanol to 33.3 mg / mL. Incubate the mixture in a 37°C metal bath for more than 20 minutes;

[0055] Preparation of aqueous phase: dilute fLuc mRNA to a final concentration of 0.17 mg / mL with 50 mM citrate buffer (pH 4.0) for later use (the fLuc mRNA sequence is shown in SEQ ID NO. 1);

[0056] The aqueous phase and the organic phase were quickly mixed at a volume ratio of 3:1 using a microfluidic device, with a total flow rate of 12 mL / min. The mixed solution was added to a dialysis bag (dialysis bag MWCO: 8000-14000) and dialyzed in 1×PBS (pH 7.4) for 18 h (ambient temperature was room temperature or 4°C) to obtain lipid nanoparticles LNP that stably encapsulated mRNA.

[0057] (b) Preparation of polymer-lipid delivery system:

[0058] Preparation of organic phase: Weigh lipids using a 1 / 100,000 balance, dissolve lipids SM102, DSPC, Cholesterol and DMG-PEG2000 in ethanol at a molar ratio of 49:20.1:30:0.9 and mix, and dilute the total lipid concentration with ethanol to 33.3 mg / mL; incubate the mixture in a 37°C metal bath for more than 20 min;

[0059] Preparation of aqueous phase: Use 50 mM citrate buffer (pH 4.0) to prepare Fluc mRNA and polymer PPG3K into 0.34 mg / mL and 13.3 mg / mL stock solutions respectively, and then mix the Fluc mRNA stock solution and the PPG3K stock solution in a volume ratio of 1:1 for standby use;

[0060] The aqueous phase and the organic phase were quickly mixed at a volume ratio of 3:1 using a microfluidic device, with a total flow rate of 12 mL / min. The mixed solution was added to a dialysis bag (dialysis bag MWCO: 8000-14000) and dialyzed in 1×PBS (pH 7.4) for 18 h (ambient temperature was room temperature or 4°C) to obtain a polymer-lipid delivery system that stably encapsulated mRNA.

[0061] (c) Preparation of physical mixture formulation (LNP+4arm PEG10K formulation)

[0062] Dilute 4arm PEG10K with 1× PBS to 15 mg / mL, filter through a 0.22 μm filter membrane, and set aside.

[0063] The LNP solution prepared and purified in the above "(a)" and the above 4arm PEG10K were mixed thoroughly at a volume ratio of 4:1. After incubation at room temperature for 20 minutes, the mixture was filtered through a 0.22 μm filter membrane and set aside.

[0064] Example 2, Particle size, PDI and encapsulation efficiency results of polymer-lipid delivery system

[0065] Preparation of LNP delivery system: The method described in "Example 1(a)" above was used.

[0066] Preparation of polymer-lipid delivery system: The polymer-lipid delivery system containing different types of polymers as shown in the figure was prepared using the method described in "Example 1(b)" above.

[0067] The particle size and PDI of polymer-lipid delivery systems and classical LNP lipid nanoparticles were determined by dynamic light scattering (DLS) at 25°C using a Zetasizer Nano ZS instrument. TM The RiboGreen RNA detection kit evaluates the effectiveness of different nanoparticles encapsulating mRNA. The specific method is as follows: Different nanoparticle samples were added to 1×TE buffer containing or not containing Triton X-100 (final concentration of 1% Triton X-100), and the content of free mRNA and total mRNA was determined. Five concentrations of mRNA standards were prepared using 1×TE buffer. The nanoparticle samples or mRNA standards were mixed with RiboGreen reagent (1:200 dilution) in a transparent bottom black plate and incubated at room temperature for 3 minutes. The fluorescence intensity was measured using an ELISA reader at an excitation wavelength of 480nm and an emission wavelength of 520nm. EE (%) = [(fluorescence of total mRNA - fluorescence of free mRNA) / fluorescence of total mRNA] × 100%.

[0068] The test results are as follows Figure 1 , Figure 2 and Figure 3 shown.

[0069] Example 3. Results of the ability of lipid nanoparticles in polymer-lipid delivery system to resist atomization shear

[0070] Preparation of LNP delivery system: The method described in "Example 1(a)" above was used.

[0071] Preparation of polymer-lipid delivery system: Using the method described in "Example 1(b)" above, the PPG3K polymer was replaced with 4-arm PEG10K, glycerol, PLA10K, ED-2003 and PLA2K-PEG5K.

[0072] The polymer-lipid formulation or LNP formulation with a dosage of 50 μg mRNA / mouse was placed in the nebulizer system with a nebulization rate of 30 μL / 10s and a nebulization time of 15 minutes. The particle size after nebulization remained at 90-110nm, with a PDI of 0.09-0.15, while the nebulization process significantly increased the particle size of LNP from 75nm to 140nm, with a PDI of 0.23. Due to the ability to resist shear force, more than 80% of the mRNA after nebulization was still encapsulated in the PPG3K, 4arm PEG10K, glycerol, PLA10K, In the preparations of ED-2003 and PLA2K-PEG5K, the mRNA encapsulation efficiency of the LNP after atomization was less than 50%, confirming that the mRNA encapsulation efficiency of the LNP based on PPG3K, 4arm PEG10K, glycerol, PLA10K, The polymer-lipid delivery system prepared by ED-2003 and PLA2K-PEG5K can withstand the shear stress generated during atomization ( Figure 4 ).

[0073] Example 4. Cellular uptake results of polymer-lipid delivery system

[0074] Preparation of LNP delivery system: The method described in "Example 1(a)" above was used.

[0075] Preparation of polymer-lipid delivery system: The polymer-lipid delivery system containing different types of polymers as shown in the figure was prepared using the method described in "Example 1(b)" above.

[0076] In the cellular uptake studies, cells were seeded at 2 × 10 5 16HBE cells were plated in 24-well plates and incubated overnight. When the cells reached about 70% density, different preparations containing 1 μg Cy5 mRNA were added to each well and incubated for 6 h. The cells were collected, washed twice with PBS, and intracellular Cy5 was further analyzed by flow cytometry. The results showed that in all the cell lines tested, the polymer-lipid delivery system group showed significantly higher uptake efficiency compared with the LNP group ( Figure 5 ).

[0077] Example 5. Cell transfection results of polymer-lipid formulations encapsulating EGFP mRNA

[0078] Preparation of LNP delivery system: The method described in "Example 1(a)" above was used.

[0079] Preparation of polymer-lipid delivery system: The polymer-lipid delivery system containing different types of polymers as shown in the figure was prepared using the method described in "Example 1(b)" above.

[0080] 1×10 5 16HBE cells / well were seeded in a 12-well plate. A confluence of 70-80% was achieved in the 12-well plate 24 h before transfection. The culture medium was aspirated and the cells were washed with 1× PBS. 170 μL of serum-free Opti-MEM was added to each well, and then 30 μL of PPG3K-lipid preparation containing 1 μg EGFP mRNA was added to each well (the sequence of EGFP mRNA is shown in SEQ ID NO.2). After 6 h of culture, the culture medium was replaced with complete culture medium, and the expression of green fluorescent protein was analyzed by flow cytometry after a further 24 h of incubation. The results showed that compared with the LNP group, the polymer-lipid preparation group encapsulating EGFP mRNA expressed more EGFP protein ( Figure 6 ).

[0081] Example 6. Transfection results of polymer-lipid formulations encapsulating MetLuc mRNA in different cells

[0082] Preparation of LNP delivery system: The method described in "Example 1(a)" above was used.

[0083] Preparation of polymer-lipid delivery system: The polymer-lipid delivery system containing different types of polymers as shown in the figure was prepared using the method described in "Example 1(b)" above.

[0084] DC 2.4 cells (1×10 5 cells / well), 16HBE cells (1.5×10 5 cells / well) and A549 cells (1.5×10 5 cells / well) and incubated overnight at 37°C 5% CO2 to promote adhesion. A density of 70-80% was reached in a 96-well plate 24h before transfection. The culture medium was aspirated and the cells were washed with 1× PBS. 170μL of serum-free Opti-MEM was added to each well, and then 30μL of polymer-lipid preparation and LNP preparation containing 400ng MetLuc mRNA (MetLuc mRNA sequence is shown in SEQ ID NO.2) were added to each well. After 6h of culture, the culture medium was replaced with complete medium and incubated for another 24h. To detect luciferase activity, 50μL of the supernatant of the transfected cells was mixed with 30μL of luciferase substrate, and the expression of luciferase activity was measured by spectrophotometer. The results are shown in Figure 7As shown, in DC 2.4 cells, 16HBE cells and A549 cells, the luciferase activity of the polymer-lipid group was significantly higher than that of the LNP group.

[0085] Example 7. Cytotoxicity test results of polymer-lipid delivery system nanomaterials

[0086] Preparation of LNP delivery system: The method described in "Example 1(a)" above was used.

[0087] Preparation of polymer-lipid delivery system: The polymer-lipid delivery system containing different types of polymers as shown in the figure was prepared using the method described in "Example 1(b)" above.

[0088] 16HBE cells were plated at 1×10 4 The cells were inoculated at a density of 100 ng / mL in a 96-well plate and cultured overnight. The next day, the cells were treated with 100 ng of mRNA@PPG3K at a fixed mRNA concentration at 37°C for 24 h. After incubation, the supernatant was removed and CCK8 solution (diluted in Opti-MEM) was added to each well. The cells were then incubated with CCK8 at 37°C for 4 h, and the absorbance was measured at 450 nm using a microplate reader. The results are shown in Figure 2. Figure 8 The results showed that the polymer-lipid preparation group had lower toxicity to cells than the LNP group.

[0089] Example 8. In vivo transfection effect of polymer-lipid delivery systems containing different polymer concentrations

[0090] Preparation of LNP delivery system: Using the method described in the above “Example 1(a)”, the molar ratio of SM-102, DSPC, cholesterol and DMG-PEG2000 was adjusted to 50:10:38.5:1.5, and N / P was 6.

[0091] Preparation of polymer-lipid delivery system: Using the method described in "Example 1(b)" above, the molar ratio of SM-102, DSPC, cholesterol and DMG-PEG2000 was adjusted to 50:10:38.5:1.5, N / P was 6, and the PPG3K polymer was replaced with 4arm PEG10K and PEG5K-b-PCL2K (the concentration of the polymer shown in the figure is the final concentration in the polymer-lipid particle).

[0092] The results showed that the polymer-lipid delivery system prepared with different concentrations of polymers could significantly increase the level of luciferase produced by Fluc mRNA in the lungs after nasal administration (in), especially the polymer-lipid formulations containing 10 mg / mL 4arm PEG10K, 5 mg / mL PPG3K and 10 mg / mL PEG5K-b-PCL2K significantly improved the transfection efficiency of mRNA in the lungs ( Fig. 9 ).

[0093] Example 9. In vivo transfection effects of polymer-lipid delivery systems containing different types of polymers

[0094] Preparation of LNP delivery system: Using the method described in the above “Example 1(a)”, the molar ratio of SM-102, DSPC, cholesterol and DMG-PEG2000 was adjusted to 50:10:38.5:1.5, and N / P was 6.

[0095] Preparation of polymer-lipid delivery system: Using the method described in "Example 1(b)" above, the molar ratio of SM-102, DSPC, cholesterol and DMG-PEG2000 was adjusted to 50:10:38.5:1.5, N / P was 6, and polymer-lipid delivery systems containing different types of polymers were prepared as shown in the figure.

[0096] The results showed that compared with LNP, hydrophilic polymers (glycerol, PEI, 4arm PEG10K and PVA27K), hydrophobic polymers (PLA10K, PPG3K, PEA2000, PCL14K and PVP15K) and amphiphilic polymers ( The polymer-lipid prepared by ED-2003, PEG2K-b-PEG5K and PLA2K-PEG5K can efficiently transfect Fluc mRNA in the lungs after nasal administration (in). Fig.10 ).

[0097] Example 10: In vivo transfection effect of physical mixed preparations administered intranasally (in)

[0098] Preparation of LNP delivery system: Using the method described in the above “Example 1(a)”, the molar ratio of SM-102, DSPC, cholesterol and DMG-PEG2000 was adjusted to 50:10:38.5:1.5, and N / P was 6.

[0099] Preparation of physical mixture formulation: The method described in "Example 1(b)" was used, except that 4arm PEG10K was replaced by PEG2K-b-PEG5K.

[0100] The results showed that the luciferase expression levels in the physical mixture preparation LNP+4arm PEG10K group and LNP+PEG2K-b-PEG5K group were significantly higher than those in the LNP preparation group ( Fig.11 ).

[0101] Example 11. In vivo transfection effect of polymer-lipid delivery systems prepared with different N / P ratios and lipid molar ratios. Preparation of LNP delivery system: The method described in the above “Example 1(a)” was used.

[0102] Preparation of polymer-lipid delivery system: Using the method described in "Example 1(b)" above, the molar ratio of SM-102, DSPC, cholesterol and DMG-PEG2000 was adjusted, for example Fig.12 shown.

[0103] The results showed that compared with LNP, polymer-lipid delivery systems containing different N / P ratios and lipid molar ratios could significantly increase the level of luciferase produced by fLuc mRNA in the lungs after nasal administration (in), especially polymer-lipid formulations with N / P ratios of 6, 8, and 10 ( Fig.12 A). In addition, the luciferase expression of the polymer-lipid delivery system containing different lipid molar ratios was significantly higher than that of LNP ( Fig.12 BD).

[0104] Example 12: In vivo transfection effects of polymer-lipid delivery systems prepared with different types of lipids

[0105] Preparation of LNP delivery system: The method described in "Example 1(a)" above was used.

[0106] Preparation of polymer-lipid delivery system: Using the method described in "Example 1(b)" above, SM-102, DSPC, cholesterol and DMG-PEG2000 were replaced by Fig.13 Lipids shown.

[0107] The results showed that compared with the LNP preparation group, the polymer-lipid preparations prepared by different lipids could significantly increase the luciferase level in the lungs after nasal administration (in). Fig.13 ).

[0108] Example 13. Tissue targeting and cell targeting results of polymer-lipid delivery system (PPG3K) after respiratory administration

[0109] The distribution of DID fluorescent-labeled preparations in the excised organs of mice 6 hours after intranasal administration (in) of polymer-lipid delivery system (PPG3K) or LNP, and the quantitative comparison of their signal intensity ( Fig. 9 , A). The proportion of cells that took up the fluorescently labeled polymer-lipid delivery system in different cells was detected by flow cytometry, where ECs represent epithelial cells, T1PCs represent type 1 pneumocytes, NTs represent neutrophils, Mφ represent macrophages, and DCs represent dendritic cells. The results are shown in Fig. 9 As shown in B. The results showed that the polymer-lipid delivery system group and the LNP group were only detected in the lungs, and no preparations were detected in the brain, heart, liver, spleen and kidneys. Compared with the LNP group, the efficiency of uptake of the polymer-lipid delivery system in the lungs was higher, especially epithelial cells, type 1 pneumocytes, macrophages and dendritic cells had a higher uptake efficiency of the polymer-lipid delivery system, while the number of neutrophils that took up the polymer-lipid delivery system was less ( Fig.14 ).

[0110] Luciferase activity in different tissues was analyzed 6 hours after intranasal administration (in) of polymer-lipid delivery system (PPG3K) or LNP encapsulating fLuc mRNA. Fig.15 As shown in A and B, luciferase activity was detected only in the lungs of both the polymer-lipid delivery system group and the LNP group, and no luciferase activity was detected in the brain, heart, liver, spleen, and kidneys. The luciferase protein expression in the lungs of the polymer-lipid delivery system group was significantly higher than that of the LNP group. Six hours after intranasal administration (in) of the polymer-lipid delivery system (PPG3K) or LNP encapsulating EGFP mRNA, the proportion of cells expressing EGFP protein in different cells was detected by flow cytometry. The results are shown in Fig.15 As shown in C, the proportion of EGFP-positive cells in epithelial cells, type 1 pneumocytes, macrophages and dendritic cells in the polymer-lipid preparation group was significantly higher than that in the LNP group, and the proportion of EGFP-positive cells in neutrophils in the polymer-lipid preparation group was significantly lower than that in the LNP group.

[0111] Example 14: Pharmacokinetic study of proteins expressed by mRNA mediated by polymer-lipid delivery system

[0112] Preparation of LNP delivery system: The method described in "Example 1(a)" above was used.

[0113] Preparation of polymer-lipid delivery system: The method described in "Example 1(b)" above was used.

[0114] The expression of luciferase was detected 6h, 12h, 24h, 48h and 72h after intranasal administration (in) of LNP and polymer-lipid preparations. The results showed that the luciferase activity of the polymer-lipid preparation group was higher than that of the LNP group at all time points, especially 6h, 12h and 24h after administration. As time went on, the luciferase expression levels of both the LNP and polymer-lipid preparation groups were the highest at 6h and 12h after administration, and the luciferase signal was significantly lower than that at 12h after administration at 24h, and almost no luciferase signal was detected at 48h and 72h after administration ( Fig.16 ).

[0115] Example 15: Safety study of polymer-lipid delivery system (PPG3K) after administration via respiratory tract

[0116] To investigate the potential toxicity of the polymer-lipid delivery system (PPG3K), lung tissue histopathology and blood liver and kidney chemistry indices were analyzed 48 h after intranasal administration of the polymer-lipid delivery system (PPG3K) or LNP. Fig.17 As shown. The results showed that no significant necrosis, edema, inflammation or neutrophil infiltration was found in the lung sections of the polymer-lipid delivery system group. However, mild peribronchial inflammation was observed in the lung sections of the LNP group). Compared with the LNP group, the blood liver and kidney chemical indicators of the polymer-lipid delivery system group were within a reasonable range, which further demonstrated the safety of the polymer-lipid delivery system.

[0117] Example 16: Study on the immune effect of mRNA vaccine based on PPG3K-lipid delivery system

[0118] Preparation of LNP delivery system: The method described in "Example 1(a)" above was used.

[0119] Preparation of polymer-lipid delivery system: The method described in "Example 1(b)" above was used.

[0120] Using mRNA encoding Klebsiella pneumoniae KP antigen as a vaccine model, the immune effects of KPmRNA / PPG3K-lipid vaccine and KPmRNA / LNP vaccine in mice were investigated by intranasal administration (in). The immunization dose was 3 μg KPmRNA / mouse, and the first vaccination time was counted as "day 0", and booster immunization was performed on day 21.

[0121] The results showed that high levels of antigen-specific IgG antibodies were detected in the serum on the 28th day after the primary immunization, and high levels of antigen-specific sIgA antibodies were detected in the bronchoalveolar lavage fluid (BALF) and nasal lavage fluid (NLF) on the 28th day after the primary immunization. Fig.18 AC). The results of bacterial load in mouse lungs showed that KPmRNA / PPG3K-lipid vaccine enhanced the killing and clearance ability of mice against K1 type Klebsiella pneumoniae ( Fig.18 D); During the infection period, the weight loss of mice in the PBS group and the LNP vaccine group was significantly greater than that in the PPG3K-lipid vaccine group; 6 days after infection, the PPG3K-lipid vaccine group had a 90% survival rate, while the LNP vaccine group had only a 50% survival rate.

[0122] The above-described embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or changes made by those skilled in the art based on the present invention are within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

1. A polymer-lipid composition capable of mediating efficient nucleic acid transfection, characterized in that: The composition comprises a polymer, a cationic lipid, a non-cationic lipid, and an active or therapeutic agent, wherein the active or therapeutic agent comprises a nucleic acid; The polymer comprises at least one of an amphiphilic polymer, a hydrophilic polymer or a hydrophobic polymer, including but not limited to: O,O′-bis(2-aminopropyl)polypropylene glycol-block-polyethylene glycol-block-polypropylene glycol, polylactic acid-co-glycolic acid, polyethylene glycol-b-polycaprolactone containing or not containing a functional group, polylactic acid-polyethylene glycol containing or not containing a functional group, polyarylether-polycaprolactone containing or not containing a functional group, polyethylene glycol-b-polycaprolactone-b-polyamino ester, polyethylene glycol-polylactic acid - One or more of glycolic acid copolymer, lauryl alcohol polyether, methacrylate copolymer, polyethylene glycol, four-arm polyethylene glycol containing or not containing functional groups, eight-arm polyethylene glycol containing or not containing functional groups, polyvinyl alcohol, polyacrylamide, branched polyethylene imine, linear polyethylene imine, glycerol and derivatives, dipropylene glycol, gelatin, hydroxypropyl methylcellulose, carbomer, chitosan and derivatives, polylactic acid, polypropylene glycol, polyetheramine, polycaprolactone, polyvinyl pyrrolidone, cellulose acetate phthalate, and acrylic resin; The nucleic acid comprises at least one selected from the group consisting of messenger RNA, self-amplifying RNA, circular RNA, small interfering RNA, short hairpin RNA and micro RNA, primary-miRNA, antisense oligonucleotides, transfer RNA, plasmid DNA, single-stranded DNA, double-stranded DNA (dsDNA), deoxyribozymes, ribozymes, nucleic acid aptamers, clustered regularly interspaced short palindromic repeats-associated nucleic acids, single guide RNA, CRISPR-RNA, trans-activating crRNA, guide RNA, single-stranded RNA and double-stranded RNA.

2. The composition according to claim 1, characterized in that: The weight percentage of the polymer in the composition is 2%-90%, or 15%-80%, or 35%-70%, or 45%-70%, or 45%-60%, or 50%-60%.

3. The composition according to claim 1, characterized in that: The cationic lipid is selected from one or more of permanent cationic lipids and ionizable cationic lipids; the molar ratio of the cationic lipid to the total lipid is 35%-65%, or 40%-60%, or 45%-55%, or 45%-50%.

4. The composition according to claims 1 and 3, characterized in that: The cationic lipid is selected from at least one of the following: DOTMA, DOSPA, DOTAP, ePC, DODAP, DODMA, DDAB, DSDMA, DODAC, DOAP, DMRIE, DOGS, DMOBA, HGT5000, HGT5001, HGT5002, HGT4001, HGT4002, HGT4003, HGT4005, DLin-MC3-DMA, DLin-KC2-DMA, Acuitas ALC-0315, Acuitas A9, Acuitas Lipid 2,2, Moderna Lipid H (SM-102), Moderna Lipid 5. A2-Iso5-2DC18, BAME-O16B, 9A1P9, C12-200, cKK-E12, OF-Deg-Lin, 306Oi10, TT3, FTT5, Lipid319, 5A2-SC8, Genevant CL1, DLinDMA, DLenDMA, ClinDMA, CpLinDMA, imidazole cholesteryl ester, RE-1, RE-2, RE-3, GL-67, 5A2-SC8, Acuitas A9, Arcturus Lipid2,2(8,8)4C CH3, OF-02, A18-Iso5-2DC18, BAME-O16B, A6, 98N12-5, L319, L343, 304O13, 306O138, 306O12B, 30 6-O12B, LP01, G0-C14, 7C1, Cephalin, Dlin-EG-DMA, DLinAP, DLin-MPZ, DLin-C-DAP, DLin-2-DMAP , Dlin-S-DMA, DLinDAP, DLin-MA, DLin-DAC, DLin-K-DMA, DLin-K-MPZ, DLin-K-DMA, DLin-K6-C4-DMA, DLin-K-C4-DMA, DLin-K-C3-DMA, CpLinDMA, DOcarbDAP, DLincarbDAP, C12-(2-3-2), Genevant Lipid CL1, XTC, ALNY-100, NC98-5 and their derivatives.

5. The composition according to claim 1, characterized in that: The non-cationic lipid is selected from one or more of neutral lipids, PEG lipids, and structural lipids.

6. The composition according to claims 1 and 5, characterized in that: The neutral lipids include at least one selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, lysophosphatidylcholine, lysophosphatidylethanolamine, phosphatidylserine, dioleoylphosphatidylserine, phosphatidylinositol, sphingomyelin, egg yolk sphingomyelin, cephalin, cardiolipin, phosphatidic acid, cerebroside, hexadecyl phosphate, distearoylphosphatidylcholine, dioleoylphosphatidylethanolamine, dioleoylphosphatidylcholine, dipalmitoylphosphatidylcholine, and dipalmitoylphosphatidylcholine. alkali, dioleoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, palmitoyloleoyl-phosphatidylcholine, palmitoyloleoyl-phosphatidylethanolamine, palmitoyloleoyl-phosphatidylglycerol, dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate, dipalmitoylphosphatidylethanolamine, dimyristoyl-phosphatidylethanolamine, distearoylphosphatidylethanolamine, monomethyl-phosphatidylethanolamine, dimethyl -phosphatidylethanolamine, di-trans oleoyl-phosphatidylethanolamine, stearoyl oleoyl-phosphatidylethanolamine, lysophosphatidylcholine, egg yolk phosphatidylcholine, dilinoleoyl phosphatidylcholine, 1,2-dipalmitoyl-sn-glycero-3-O-4'-(N,N,N-trimethyl)-homoserine, monogalactosyl diacylglycerol, diacetyl diacylglycerol, sulfaquinoline diacylglycerol, 1-palmitoyl-2-cis-9,10-methylenehexyl-decanoyl-sn-glycero-3-phosphocholine, 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, MSPC, DMPC, DLPC, DPPS, DUPC and their derivatives; the molar ratio of the neutral lipids to the total lipids is 5%-35%, or 10%-25%, or 10%-20%, or 15%-20%.

7. The composition according to claims 1 and 9, characterized in that: The PEG lipid is selected from one or more of PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DSPE, ceramide-PEG2000, ALC-0159, DSPE-PEG, and PEG-PE; the molar ratio of the PEG lipid to the total lipid is 0%-5%, or 0%-3%, or 0%-1.5%, or 0%-1%, or 0.3%-1%, or 0.5%-1%.

8. The composition according to claims 1 and 9, characterized in that: The structural lipids are selected from one or more of cholesterol, coprosterol, sitosterol, ergosterol, campesterol, fucoxanthin, β-sitosterol, stigmasterol, brassicasterol, tomatine, tomatine, ursolic acid, and α-tocopherol; the molar ratio of the structural lipids to the total lipids is 20%-45%, or 25%-40%, or 25%-35%, or 25%-30%.

9. The composition according to claim 1, characterized in that The composition preparation method comprises the following steps: 1) adding the polymer to a solution containing the nucleic acid and / or a solution containing the lipid, and 2) mixing the solution containing nucleic acid and the solution containing lipid to form the composition; Or the method comprises: 1) mixing the solution containing the nucleic acid and the solution containing the lipid to form lipid nanoparticles encapsulating the nucleic acid, and 2) adding the polymer to the lipid nanoparticle solution loaded with the nucleic acid to form the composition; Or the method comprises: 1) preforming the lipid and polymer into nucleic acid polymer-lipid nanoparticles; and 2) mixing a solution comprising the nucleic acid with the polymer-lipid nanoparticle solution to form the composition; or the method comprises: 1) in a solution comprising the lipid, allowing the lipid to pre-form nucleic acid-free lipid nanoparticles; and 2) mixing the solution comprising the nucleic acid and the polymer with the lipid nanoparticle solution to form the composition.

10. The composition according to claim 1, characterized in that The polymer-lipid composition for delivering mRNA is delivered and administered via the respiratory tract, or orally, or intramuscularly, or intravenously, or subcutaneously, or intratumorally.

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