Preparation method and application of fluorinated lipid nanoparticles

Abstract

The present invention relates to the technical field of nanopharmaceuticals, and in particular to a preparation method and application of fluorinated lipid nanoparticles. The preparation method of the fluorinated lipid nanoparticles comprises: dissolving fluorinated lipids and four commercial lipid molecules in anhydrous ethanol to obtain an ethanol phase lipid solution; dissolving nucleic acid in a citric acid buffer to obtain an aqueous phase nucleic acid solution; then uniformly mixing the ethanol phase lipid solution and the aqueous phase nucleic acid solution at a ratio of 1:3, and purifying the mixture through a desalting chromatography column to obtain the fluorinated lipid nanoparticles of the present invention. Compared with commercial transfection reagents and conventional lipid nanoparticles, the nanoparticles can significantly improve their nucleic acid delivery performance and transfection efficiency.

Description

Technical Field

[0001] The present invention relates to the technical field of nanopharmaceuticals, and in particular to a preparation method and application of fluorine-containing lipid nanoparticles. Background Art

[0002] Nucleic acid is one of the important components of organisms in their life activities. After exogenous nucleic acids enter cells, they can express encoded proteins for the treatment of diseases or as vaccines to activate the body's immune system. However, exogenous nucleic acids are easily degraded by nucleases that are widely present in the body, making it difficult to exert their biological effects (K. Paunovska, D. Loughrey and JEDahlman, Nat. Rev. Genet., 2022, 23, 265–280.). Secondly, nucleic acid molecules are hydrophilic and have a strong negative charge on the surface, which makes it difficult for them to cross the cell membrane. Therefore, nucleic acid drugs need to rely on carriers to give them stability and deliver them to cells to play a role. Lipid nanoparticles (LNP) are one of the most widely used nucleic acid carriers. The two new crown nucleic acid vaccines developed by Moderna and Pfizer / BioNTech have demonstrated the excellent efficacy and good safety of LNP carriers.

[0003] LNP carriers are composed of ionizable lipids, auxiliary lipids, cholesterol and polyethylene glycol (PEG) lipids. Among these components, the chemical structure of ionizable lipids significantly affects the delivery efficiency of nucleic acids. Ionizable lipids are positively charged in acidic buffer solutions and interact with negatively charged nucleic acid molecules to form LNPs. Under physiological pH conditions, LNPs are in an uncharged state, which can avoid rapid clearance from systemic circulation. When LNPs enter the cell lysosomes, the ionizable lipids are converted back to a negatively charged state to promote the release of nucleic acids (KJ Kauffman, JR Dorkin, JHYang, MW Heartlein, F. Derosa, FFMir, OS Fenton and DG Anderson, Nano Lett., 2015, 15 (11), 7300–7306.). Although a variety of ionizable lipids with low toxicity and immunogenicity have been developed, it is still necessary to develop new ionizable lipids with higher delivery efficiency.

[0004] Studies have found that fluorinated lipids, dendritic lipids and polymers can improve the ability to deliver nucleic acids. This is mainly due to the unique properties of fluorinated lipids. Compared with hydrocarbons, fluorocarbons are hydrophobic, lipophobic and have better stability, which makes them easier to self-assemble. Studies have shown that fluorinated lipids or polymers can form more stable LNPs, thereby preventing the degradation of encapsulated nucleic acids to prolong blood circulation time (Z. Zhang, W. Shen, J. Ling, Y. Yan, J. Hu and Y. Cheng, Nat. Commun., 2018, 9, 1377.). Therefore, the development of new fluorinated ionizable lipids may be an effective strategy to improve the efficiency of LNP nucleic acid delivery. Summary of the invention

[0005] Based on the above situation, one of the objects of the present invention is to provide a series of methods for preparing fluorinated lipid nanoparticles, and the second object of the present invention is to provide the application of the fluorinated LNP for improving the efficiency of nucleic acid delivery.

[0006] The present invention provides a method for preparing fluorinated lipid nanoparticles, comprising the following steps:

[0007] Step (1): Preparation of ethanol phase lipid solution: dissolving fluorinated lipid, ionizable lipid, auxiliary lipid, cholesterol and polyethylene glycol lipid in anhydrous ethanol, and mixing them evenly to obtain ethanol phase lipid solution;

[0008] The ionizable lipid is heptadecan-9-yl-8-((2-hydroxyethyl)(6-oxo-6-((undecanyloxy)hexyl)amino)octanoate) or 4-(N,N-dimethylamino)butyric acid (dilinoleyl)methyl ester or ((4-hydroxybutyl)azadiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate);

[0009] Step (2): Preparation of an aqueous nucleic acid solution: dissolving nucleic acid molecules in a citric acid buffer to obtain an aqueous nucleic acid solution;

[0010] Step (3): Preparation of fluorinated lipid nanoparticles: mixing the ethanolic lipid solution prepared in step (1) with the aqueous nucleic acid solution prepared in step (2), and purifying the mixture using a desalting chromatography column. The purified product is fluorinated lipid nanoparticles (whose hydrated particle size is 10 to 500 nm);

[0011] The structural formula of the fluorinated lipid in step (1) is as follows:

[0012] Wherein Rf is selected from perfluorotert-butyloxy Trifluoroethoxy Difluoroethoxy Pentafluorothiobenzyloxy Monofluorobenzyloxy difluorobenzyloxy Isofluorobenzyloxy, bis(trifluoromethylbenzyloxy) trifluoromethoxybenzyloxy Trifluoromethylthiobenzyloxy and O(CH2)2(CF2) m Any of the linear polyfluoroalkyl groups of CF3 structure, m=a natural number of 1-10, and n=a natural number of 3-20.

[0013] Further, the ionizable lipid, auxiliary lipid, cholesterol and polyethylene glycol lipid are any one of the following formulas;

[0014] Formula 1: Heptadecan-9-yl-8-((2-hydroxyethyl)(6-oxo-6-((undecanyloxy)hexyl)amino)octanoate), distearoylphosphatidylcholine, cholesterol and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000;

[0015] Formula 2: 4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester, distearoyl phosphatidylcholine, cholesterol and 1,2-dimyristoyl-rac-glycero-3-methoxy polyethylene glycol 2000;

[0016] Formula 3: ((4-hydroxybutyl)azadiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), distearoylphosphatidylcholine, cholesterol and methoxypolyethylene glycol ditetradecyl acetamide.

[0017] Furthermore, the method for preparing the fluorinated lipid described in step (1) comprises the following steps:

[0018] Step (a): in the presence of a base, converting the fluorinated alcohol into the corresponding potassium salt or sodium salt to obtain the fluorinated nucleophile 1;

[0019] Step (b): a fluorinated nucleophilic reagent 1 undergoes a nucleophilic substitution reaction with a monohalogenated alkyl alcohol to obtain a fluorinated alkyl alcohol 2;

[0020] Step (c): adding an activator and an acid-binding agent 1 to the fluorinated alkyl alcohol 2 for activation to obtain an activated fluorinated alkyl alcohol 3;

[0021] Step (d): in the presence of an acid-binding agent 2 and a catalyst 1, reacting the activated fluorinated alkyl alcohol 3 with benzylamine to obtain a benzyl-protected fluorinated lipid 4;

[0022] Step (e): Finally, under a hydrogen atmosphere and catalyst 2, the benzyl protecting group in the benzyl-protected fluorinated lipid 4 is removed to obtain a fluorinated lipid. The synthetic route is as follows:

[0023]

[0024] Furthermore, the fluorine-containing alcohol in step (a) is any one of perfluoro-tert-butyl alcohol, trifluoroethanol, difluoroethanol, pentafluorothiobenzyl alcohol, fluorobenzyl alcohol, bistrifluoromethylbenzyl alcohol, trifluoromethoxybenzyl alcohol, and trifluoromethylthiobenzyl alcohol, and the base is sodium hydroxide or potassium hydroxide; the molar ratio of the base to the fluorine-containing alcohol is 1:1.

[0025] Furthermore, in step (b), the molar ratio of the fluorine-containing nucleophilic reagent 1 to the monohalogenated alkyl alcohol is 1.0:(1.1-3.0), and the monohalogenated alkyl alcohol is 12-bromo-1-dodecanol; the nucleophilic substitution reaction temperature is 80-120° C., and more preferably the reaction is stirred at 110° C. for 12 hours;

[0026] Furthermore, in step (c), p-toluenesulfonyl chloride is used as an activating agent, triethylamine or N,N-diisopropylethylamine is used as an acid-binding agent 1, the activation temperature is 0°C, and the molar ratio of the fluorinated alkyl alcohol 2 to the activating agent and the acid-binding agent 1 is 1.0:(1.5-3.0):(2.0-4.0), preferably 1:2:2;

[0027] Further, in step (d), potassium carbonate is used as the acid-binding agent 2, potassium iodide is used as the catalyst 1, the molar ratio of benzylamine to the activated fluorinated alkyl alcohol 3, the acid-binding agent 2, and the catalyst 1 is 1.0:(2.0-4.0):(3.0-6.0):1.0, preferably 1.0:2.4:4.0:1.0, and the reaction temperature is 85° C. and refluxed until the reaction is complete;

[0028] Furthermore, in step (e), 10% palladium carbon or palladium hydroxide is used as catalyst 2, the reaction is carried out under a hydrogen atmosphere, the reaction solvent is methanol, and the molar ratio of the benzyl-protected fluorinated lipid 4 to the active substance in catalyst 2 is 1.0:(0.1-0.4).

[0029] Furthermore, the mass ratio of the fluorinated lipids in step (1) to the total mass of the fluorinated lipids and the ionizable lipids is between 1% and 100%, preferably between 50% and 75%, and most preferably 50%.

[0030] Furthermore, the mass ratio of the fluorinated lipid, ionizable lipid, auxiliary lipid, cholesterol and polyethylene glycol lipid described in step (1) is 1:(1-3):(0.44-0.88):(0.8-1.6):(0.21-0.43).

[0031] Furthermore, the nucleic acid molecule described in step (2) is any one of plasmid DNA, mRNA, and siRNA, the pH of the citric acid solution is 3, and the concentration of nucleic acid in the aqueous nucleic acid solution is less than 1 mg / mL.

[0032] Furthermore, the volume ratio of the ethanol phase lipid solution to the aqueous phase nucleic acid solution in step (3) is 1:(0.1-10), preferably 1:3.

[0033] The present invention also provides an application of fluorinated lipid nanoparticles obtained by the above preparation method in a nucleic acid delivery vector, which can significantly improve the nucleic acid transfection efficiency and protein expression level.

[0034] Compared with the prior art, the advantages and beneficial effects of the present invention are:

[0035] The fluorinated lipid nanoparticles prepared by the preparation method of the present invention can significantly improve the transfection efficiency of nucleic acids by adjusting the ratio of fluorinated lipids to ionizable lipids, which may be due to the fact that the fluorinated lipid nanoparticles are more stable, have better cell uptake effects and more significant lysosomal escape effects. The protein expression level of the fluorinated lipid nanoparticle group is better than that of the commercial transfection reagent group and the non-fluorinated lipid nanoparticle group. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Schematic diagram of the principle of the test experiment in Example 3.

[0037] Figure 2-Figure 4 The di(12-(perfluorotert-butoxy)dodecyl)amine prepared in Example 1 1 H NMR, 13 C NMR and 19 F NMR spectrum.

[0038] Figure 5 This is the TEM image of the fluorinated lipid nanoparticles prepared in Example 2.

[0039] Figure 6 This is the DLS graph of the fluorinated lipid nanoparticles prepared in Example 2.

[0040] Figure 7 The fluorescence intensity and encapsulation efficiency test results of the lipid nanoparticles prepared in Example 3.

[0041] Figure 8 This is the confocal result of transfecting 293T cells with lipid nanoparticles prepared in Example 3.

[0042] Fig. 9 for Figure 8 Quantitative analysis of fluorescence intensity in the CCP images. DETAILED DESCRIPTION

[0043] The technical solution of the present invention is described in detail below in conjunction with specific embodiments and drawings.

[0044] The main reagents and materials used in the following examples are described as follows: Heptadecan-9-yl-8-((2-hydroxyethyl)(6-oxo-6-((undecyloxy)hexyl)amino)octanoate) was purchased from Xiamen Sinopang, distearoylphosphatidylcholine and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000 were purchased from Ruixi Biotechnology, and cholesterol was purchased from Sigma. PD-10 desalting chromatography column (Cat. No. 17085101) was purchased from GE. Ulysis TM Alexa Fluor TM 647 Nucleic Acid Labeling Kit (Cat. No. U2660) was purchased from Thermo Fisher. 293T cells were purchased from Shanghai Cell Bank, Chinese Academy of Sciences. Lipo8000 (Cat. No. C0533) and DAPI stain (Cat. No. C1005) were purchased from Bio-Tech. Plasmid Extraction Kit (Cat. No. DP116) was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0045] Example 1 Preparation and characterization of di(12-(perfluorotert-butoxy)dodecyl)amine, the specific steps are as follows:

[0046]

[0047] Step a: Dissolve 63.5 mmol of potassium hydroxide in 25 mL of water, cool to 0°C, slowly add 63.5 mmol of perfluoro-tert-butanol, stir at 0°C for 1 hour, remove water by freeze drying, and obtain potassium perfluoro-tert-butoxide as a white solid (17.4 g, 99% yield).

[0048]

[0049] Step b: Dissolve 52.2mmol of potassium perfluorotert-butoxide in 150mL of N,N-dimethylformamide (DMF), and then add 62.7mmol of 12-bromo-1-dodecanol. Stir the resulting reaction solution at 110°C for 12h. After the reaction is complete, remove DMF by distillation under reduced pressure. Dissolve the crude product in 200mL of dichloromethane, wash with 100mL of water, separate the dichloromethane layer, and wash with 100mL of water. Finally, dry the organic phase with anhydrous sodium sulfate, filter, and purify the filtrate by column after drying to obtain fluorinated alkyl alcohol 2, which is a colorless oily liquid (17.2g, 78% yield). 1 H NMR (500MHz, CDCl3) δ3.98(t,J=6.5Hz,2H),3.63(t,J=6.7Hz,2H),1.70–1.61(m,2H),1.59–1.51(m,2H),1.40–1.22(m,16H).

[0050]

[0051] Step c: Add 2.4 mmol of fluorinated alkyl alcohol 2 to a reaction flask, dissolve it with dichloromethane (DCM, 10 mL), and then add triethylamine (Et3N, 0.66 mL, 4.8 mmol). After stirring at 0°C for 10 min, add 10 mL of a DCM solution of p-toluenesulfonyl chloride (TsCl, 4.8 mmol) dropwise, and continue the reaction for 5 h. After the reaction is complete, the reaction solution is extracted three times with DCM, and then washed three times with saturated ammonium chloride. The combined organic phase is dried over anhydrous sodium sulfate and filtered. The filtrate is spin-dried and purified by column to obtain the activated fluorinated alkyl alcohol 3, which is a colorless oily liquid (1.1 g, 81% yield). 1 H NMR (500MHz, CDCl3) δ7.78(d,J=8.4Hz,2H),7.33(d,J=8.0Hz,2H),4.00(dt,J=12.1,6.5 Hz, 4H), 2.43 (s, 3H), 1.69–1.59 (m, 4H), 1.35 (q, J = 7.2, 6.8Hz, 2H), 1.32–1.17 (m, 14H). 13 C NMR (126MHz, CDCl3) δ144.8,139.3,133.4,129.9,128.0,124.1,120.6(t,J=294.2Hz),114.2,80 .4–79.2(m),70.7,69.8,33.8,29.7,29.42,29.40,29.38,29.3,29.1,28.9,28.8,25.30,25.26. 19 F NMR(471MHz,CDCl3)δ-73.48.HRMS m / z:[M+Na] + Calculate for C 23 H 31 F9NaO4S,597.1692,Found597.1691.

[0052]

[0053] Step d: Add potassium carbonate (K2CO3, 2.9mmol) and potassium iodide (KI, 0.73mmol) to a two-necked round-bottom flask, and then replace nitrogen three times. Then add benzylamine (0.73mmol) and activated fluorinated alkyl alcohol 3 (0.87mmol) in anhydrous acetonitrile (CH3CN, 10mL) solution in turn. Then place the reaction solution in an 85°C oil bath for reflux reaction for 6h; then add activated fluorinated alkyl alcohol 3 (0.87mmol) in anhydrous CH3CN (10mL) solution again, and continue to reflux reaction for 6h. After the reaction is complete, dissolve the crude product with 100mL of ethyl acetate, then wash twice with 100mL of water, and finally dry the organic phase with anhydrous sodium sulfate, filter, and purify the filtrate by column after spin drying to obtain benzyl-protected fluorinated lipid 4, which is a colorless oily liquid (599.8mg, 91% yield). 1 H NMR (500MHz, CDCl3) δ7.33(dt,J=14.9,7.5Hz,4H),7.24(t,J=7.2Hz,1H),4.02(t,J=6.5Hz,4H),3.57(s,2H),2.42(t, J=7.4Hz,4H),1.69(p,J=6.7Hz,4H),1.48(qd,J=8.0,5.2,3.7Hz,4H),1.40(dd,J=9.2,5.4Hz,4H),1.36–1.26(m,28H). 13 C NMR (126MHz, CDCl3) δ140.3,139.2,128.8,128.0,126.6,120.4(t,J=294.2Hz),114.0,80.4–79.2(m) ,69.8,58.7,53.8,33.8,32.0,29.7,29.65,29.59,29.54,29.46,29.2,29.0,27.5,27.0,25.3,22.7. 19 F NMR (471 MHz, CDCl3) δ-73.54.

[0054]

[0055] Step e: Add the benzyl-protected fluorinated lipid 4 (1.1 mmol) and 10% palladium carbon (Pd / C, mass fraction, containing 0.22 mmol Pd) to the reaction bottle, add 10 mL of anhydrous methanol to dissolve, then replace the hydrogen gas in the reaction system three times, and insert a hydrogen balloon to react for 12 hours. After the reaction is complete, the reaction solution is filtered, the filtrate is dried and then purified by column to obtain the fluorinated lipid 5, named di(12-(perfluorotert-butoxy)dodecyl)amine, which is a colorless oily liquid (720.9 mg, 80% yield). 1HNMR(500MHz, CDCl3)δ3.97(t,J=6.5Hz,4H),2.62–2.57(m,4H),1.65(d,J=6.6H z, 4H), 1.50 (d, J = 7.4Hz, 4H), 1.35 (t, J = 7.6Hz, 4H), 1.26 (q, J = 4.8, 3.7Hz, 28H). 13 C NMR (126MHz, CDCl3) δ120.4 (t, J = 294.8Hz), 80.4–79.4 (m), 69.8, 49.9, 31.9, 29.8, 29.7, 29.54, 29.52, 29.47, 29.4, 29.3, 29.1, 27.4, 25.2, 22.7. 19 FNMR (471MHz,CDCl3)δ-73.53.

[0056] Example 2 Preparation and characterization of fluorinated lipid nanoparticles, the specific steps are as follows:

[0057] (1) 1.5 mg of heptadecan-9-yl-8-((2-hydroxyethyl)(6-oxo-6-((undecanyloxy)hexyl)amino)octanoate, 0.33 mg of distearoylphosphatidylcholine, 0.6 mg of cholesterol and 0.16 mg of 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000 were dissolved in 405.3 μL of anhydrous ethanol and mixed well to obtain a fluorine-free ethanol phase lipid solution.

[0058] (2) 0.75 mg of 9-heptadecan-8-((2-hydroxyethyl)(6-oxo-6-((undecanyloxy)hexyl)amino)octanoate), 0.75 mg of di(12-(perfluoro-tert-butyloxy)dodecyl)amine, 0.33 mg of distearoylphosphatidylcholine, 0.6 mg of cholesterol and 0.16 mg of 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000 were dissolved in 405.3 μL of anhydrous ethanol and mixed to obtain a fluorinated ethanol phase lipid solution.

[0059] (3) The pEGFP plasmid was extracted from the Escherichia coli expressing the pEGFP plasmid using a plasmid extraction kit. 0.15 mg of the pEGFP plasmid was dissolved in 1283.8 μL of 50 mM citric acid buffer at pH 3 to obtain an aqueous nucleic acid solution.

[0060] (4) The fluorine-free ethanol phase lipid solution and the aqueous phase nucleic acid solution are uniformly mixed at a volume ratio of 1:3 to obtain fluorine-free lipid nanoparticles (pEGFP-LNP-SM102).

[0061] (5) The fluorinated ethanol phase lipid solution and the aqueous phase nucleic acid solution were uniformly mixed at a volume ratio of 1:3 to obtain fluorinated lipid nanoparticles (pEGFP-LNP-F12C).

[0062] (6) PD-10 was used to purify pEGFP-LNP-SM102 and pEGFP-LNP-F12C to remove unreacted lipids and citric acid to obtain purified fluorine-free lipid nanoparticles pEGFP-LNP-SM102 and fluorine-containing lipid nanoparticles pEGFP-LNP-F12C.

[0063] (7) The appearance of purified pEGFP-LNP-SM102 and pEGFP-LNP-F12C was observed using a transmission electron microscope (TEM), as follows: the sample was dissolved in water, a drop of the sample solution was dropped on a copper mesh, excess liquid was absorbed with filter paper, and after drying, a drop of 1% phosphotungstic acid was dropped on the copper mesh. After the sample was dried, it was observed under a transmission electron microscope. The results are shown in FIG. Figure 5 As shown, pEGFP-LNP-SM102 and pEGFP-LNP-F12C are spherical. DLS showed that the hydrated particle sizes of pEGFP-LNP-SM102 and pEGFP-LNP-F12C were 248.8 nm and 257.6 nm, respectively. Figure 6 ).

[0064] Example 3 Detection of nucleic acid encapsulation efficiency of fluorinated lipid nanoparticles, the specific steps are as follows:

[0065] (1)Using Ulysis TM Alexa Fluor TM pEGFP was linked to Cy5 using the 647 Nucleic Acid Labeling Kit to obtain pEGFP-Cy5.

[0066] (2) 150 μg of 9-heptadecan-8-((2-hydroxyethyl)(6-oxo-6-((undecanyloxy)hexyl)amino)octanoate), 33 μg of distearoylphosphatidylcholine, 60 μg of cholesterol and 16 μg of 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000 were dissolved in 40.5 μL of anhydrous ethanol and mixed well to obtain a fluorine-free ethanol phase lipid solution.

[0067] (3) 112.5 μg of 9-heptadecan-8-((2-hydroxyethyl)(6-oxo-6-((undecanyloxy)hexyl)amino)octanoate), 37.5 μg of di(12-(perfluorotert-butyloxy)dodecyl)amine, 33 μg of distearoylphosphatidylcholine, 60 μg of cholesterol and 16 μg of 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000 were dissolved in 40.5 μL of anhydrous ethanol and mixed well to obtain a 25% fluorinated ethanol phase lipid solution.

[0068] (4) 75 μg of 9-heptadecan-8-((2-hydroxyethyl)(6-oxo-6-((undecanyloxy)hexyl)amino)octanoate), 75 μg of di(12-(perfluorotert-butyloxy)dodecyl)amine, 33 μg of distearoylphosphatidylcholine, 60 μg of cholesterol and 16 μg of 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000 were dissolved in 40.5 μL of anhydrous ethanol and mixed well to obtain a 50% fluorinated ethanol phase lipid solution.

[0069] (5) Take 14.85 μg of pEGFP plasmid and 0.15 μg of pEGFP-Cy5 and dissolve them in 128.4 μL of 50 mM citric acid buffer at pH = 3 to obtain an aqueous nucleic acid solution containing Cy5.

[0070] (6) Add the fluorine-free ethanol phase lipid solution to the Cy5-containing aqueous nucleic acid solution obtained in (5) and mix them at a uniform speed. The volume ratio of the fluorine-free ethanol phase lipid solution to the Cy5-containing aqueous nucleic acid solution is 1:3 to obtain fluorine-free lipid nanoparticles (pEGFP-Cy5-LNP-SM102).

[0071] (7) Add 25% fluorinated ethanol lipid solution to the Cy5-containing aqueous nucleic acid solution obtained in (5) and mix at a uniform speed. The volume ratio of the fluorinated lipid solution to the Cy5-containing aqueous nucleic acid solution is 1:3 to obtain fluorinated lipid nanoparticles (pEGFP-Cy5-LNP-25% F12C).

[0072] (8) Add 50% fluorinated ethanol lipid solution to the Cy5-containing aqueous nucleic acid solution obtained in (5) and mix at a uniform speed. The volume ratio of the fluorinated ethanol lipid solution to the Cy5-containing aqueous nucleic acid solution is 1:3 to obtain fluorinated lipid nanoparticles (pEGFP-Cy5-LNP-50%F12C).

[0073] (9) pEGFP-Cy5-LNP-SM102, pEGFP-Cy5-LNP-25%F12C and pEGFP-Cy5-LNP-50%F12C were purified using a 100 kDa ultrafiltration tube to remove unreacted nucleic acids. The filtrates were transferred to new 1.5 mL EP tubes, and supplemented to 100 μL with PBS (pH = 7.0, 0.1 M), respectively, and recorded as SM102 filtrate, 25%F12C and 50%F12C filtrate, and stored at 4°C for subsequent experiments.

[0074] (10) Figure 7 As shown, the fluorescence intensity values ​​of the three filtrates at 670 nm were detected using a fluorescence spectrometer to calculate the LNP encapsulation efficiency. The calculation formula is: The encapsulation efficiency of pEGFP-LNP-SM102 was calculated to be 74.6%, the encapsulation efficiency of pEGFP-LNP-25%F12C was 81.0%, and the encapsulation efficiency of pEGFP-LNP-50%F12C was 76.2%.

[0075] (11) Using the LNP encapsulation efficiency obtained in step (10), calculate the pEGFP concentration in the purified lipid nanoparticles obtained in step (9). The calculation formula is: The calculated pEGFP concentration of pEGFP-LNP-SM102 was 0.112 μg / μL, the pEGFP concentration of pEGFP-LNP-25%F12C was 0.122 μg / μL, and the pEGFP concentration of pEGFP-LNP-50%F12C was 0.114 μg / μL.

[0076] Test experiment: By adjusting the ratio of fluorinated lipids to ionizable lipids in fluorinated lipid nanoparticles, the transfection efficiency of nucleic acids can be significantly improved.

[0077] Test method:

[0078] The purified lipid nanoparticles prepared in step (9) of Example 3 were used to perform a transfection experiment on 293T cells. Specifically, an appropriate amount of 293T cells were seeded in a glass-bottomed confocal dish with a diameter of 2 cm and waited for them to adhere to the wall (divided equally into a control group, a fluorine-free lipid nanoparticle group, a 25% fluorine-containing lipid nanoparticle group, and a 50% fluorine-containing lipid nanoparticle group). After 12 hours, pEGFP was transfected in the control group using a commercial transfection reagent: 0.39 μg of pEGFP, 0.628 μL of Lipo8000, and 19.6 μL of serum-free DMEM culture medium were added to the confocal dish; in the fluorine-free lipid nanoparticle group, 3.5 μL of pEGFP-LNP-SM10 prepared in Example 3 was added to each well. 2 (wherein the pEGFP content is 0.39 μg) and 16.1 μL serum-free DMEM medium; in the 25% fluorinated lipid nanoparticle group, 3.2 μL of pEGFP-LNP-25%F12C prepared in Example 3 (wherein the pEGFP content is 0.39 μg) and 16.4 μL serum-free DMEM medium were added to each well; in the 50% fluorinated lipid nanoparticle group, 3.4 μL of pEGFP-LNP-50%F12C prepared in Example 3 (wherein the pEGFP content is 0.39 μg) and 16.2 μL serum-free DMEM medium were added to each well. After the cells were incubated at 37°C and 5% CO2 for 48 hours, the culture medium was removed, the cells were fixed with paraformaldehyde, and the cell nuclei were stained with DAPI stain. Fluorescence visualization was then captured using a fluorescence confocal microscope.

[0079] Test results:

[0080] like Figure 8 and Fig. 9 As shown, the EGFP protein expression of the fluorinated lipid nanoparticle group was significantly stronger than that of the commercial transfection reagent group and the non-fluorinated lipid nanoparticle group. Among them, the transfection efficiency of pEGFP-LNP-50%F12C was significantly better than that of the other three groups, and its EGFP protein expression was 26.7 times that of the commercial transfection reagent, 10 times that of the non-fluorinated nanoparticle group, and 4.4 times that of the pEGFP-LNP-25%F12C group. The experimental results show that the transfection efficiency of nucleic acids can be significantly improved by optimizing the proportion of fluorinated lipids in fluorinated nanoparticles.

Claims

1. A method for preparing fluorinated lipid nanoparticles, comprising the following steps: Step (1): Preparation of ethanol phase lipid solution: dissolving fluorinated lipid, ionizable lipid, auxiliary lipid, cholesterol and polyethylene glycol lipid in anhydrous ethanol, and mixing them evenly to obtain ethanol phase lipid solution; The ionizable lipid is heptadecan-9-yl-8-((2-hydroxyethyl)(6-oxo-6-((undecanyloxy)hexyl)amino)octanoate) or 4-(N,N-dimethylamino)butyric acid (dilinoleyl)methyl ester or ((4-hydroxybutyl)azadiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate); Step (2): Preparation of an aqueous nucleic acid solution: dissolving nucleic acid molecules in a citric acid buffer to obtain an aqueous nucleic acid solution; Step (3): Preparation of fluorinated lipid nanoparticles: mixing the ethanolic lipid solution prepared in step (1) with the aqueous nucleic acid solution prepared in step (2), and purifying the mixture using a desalting chromatography column, wherein the purified product is fluorinated lipid nanoparticles; The structural formula of the fluorinated lipid in step (1) is as follows: Wherein Rf is perfluorotert-butyloxy n=12.

2. The preparation method according to claim 1, characterized in that: The ionizable lipids, auxiliary lipids, Cholesterol and polyethylene glycol lipids are any of the following formulations; Formula 1: Heptadecan-9-yl-8-((2-hydroxyethyl)(6-oxo-6-((undecyloxy)hexyl)amino)octanoate), distearoylphosphatidylcholine, cholesterol and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000; Formula 2: 4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester, distearoyl phosphatidylcholine, cholesterol and 1,2-dimyristoyl-rac-glycero-3-methoxy polyethylene glycol 2000; Formula 3: ((4-hydroxybutyl) azadiyl) bis(hexane-6,1-diyl) bis(2-hexyldecanoate), distearyl Phosphatidylcholine, cholesterol, and methoxypolyethylene glycol ditetradecyl acetamide.

3. The preparation method according to claim 1, characterized in that: The mass ratio of the fluorinated lipids in step (1) to the total amount of the fluorinated lipids and the ionizable lipids is between 1% and 100%.

4. The preparation method according to claim 1, characterized in that: The nucleic acid molecule in step (2) is any one of plasmid DNA, mRNA, and siRNA; and / or The pH of the citric acid solution is 3, and the concentration of nucleic acid in the aqueous nucleic acid solution is less than 1 mg / mL.

5. The preparation method according to claim 1, characterized in that: The volume ratio of the ethanol phase lipid solution to the aqueous phase nucleic acid solution in step (3) is 1:(0.1-10).

6. Use of the fluorinated lipid nanoparticles obtained by the preparation method according to any one of claims 1 to 5 in the preparation of nucleic acid delivery carrier drugs.

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