A mitochondrial-targeted fluorinated ionizable lipid and its use as a mitochondrial gene delivery vector

Abstract

The present invention discloses a mitochondrially targeted fluorinated ionizable lipid and its use as a mitochondrial gene delivery vector. A mitochondrially targeted fluorinated ionizable lipid is a compound of formula (I), or a pharmaceutically acceptable salt, stereoisomer, or tautomer thereof, wherein: a = an integer from 2 to 8, b = an integer from 4 to 8; and R1 is an ionizable lipid head group, comprising a mono- or polyamine containing a primary or tertiary amine structure. A mitochondrially targeted lipid carrier comprises the fluorinated ionizable lipid described herein, a cationic lipid, an auxiliary lipid, a structural lipid, and a functional polymer conjugated lipid modified with a mitochondrial targeting group. The fluorinated ionizable lipid gene delivery vector with mitochondrial targeting capability provided by the present invention has excellent biocompatibility, can simultaneously increase the cellular uptake and mitochondrial accumulation of gene drugs, improve the efficiency of mitochondrial gene transfection, and achieve efficient delivery of gene drugs to the mitochondrial matrix. #imgabs0#

Description

Technical Field

[0001] The present invention belongs to the technical field of biopharmaceuticals, and in particular relates to a mitochondrial-targeted fluorinated ionizable lipid and its application as a mitochondrial gene delivery vector. Background Art

[0002] Gene therapy for mitochondrial gene mutation disorders such as Leber hereditary optic neuropathy (LHON) is primarily categorized into ectopic expression via viral vectors and in situ mitochondrial gene therapy via non-viral vectors. While viral vector-mediated gene therapy has made some progress in clinical research for mitochondrial-related diseases, potential safety concerns and immunogenicity risks limit its widespread application in this field. In situ mitochondrial gene therapy can deliver therapeutic genes directly to the mitochondrial matrix; therefore, non-viral vector-mediated in situ mitochondrial gene therapy may be an effective strategy to overcome the current bottlenecks in LHON gene therapy. However, the complex mitochondrial bilayer structure complicates in situ gene delivery, resulting in limited gene transfection efficiency in existing mitochondrial gene delivery systems. Therefore, to achieve efficient and low-toxic mitochondrial gene therapy, it is crucial to develop novel non-viral mitochondrial gene delivery vectors for the treatment of mitochondrial gene mutation-related diseases such as LHON. Summary of the Invention

[0003] The present invention provides a fluorinated ionizable lipid with mitochondrial targeting capability and use of the lipid as a mitochondrial gene delivery vector for treating diseases caused by mitochondrial gene mutations.

[0004] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0005] A mitochondrial-targeted fluorinated ionizable lipid, which is a compound of formula (I) or pharmaceutically acceptable salts, stereoisomers, or tautomers thereof, wherein: a=an integer of 2 to 8, b=an integer of 4 to 8; and R1 is an ionizable lipid head group, a mono- or poly-amine containing a primary or tertiary amine structure.

[0006] Preferably, the compound has one of the structures shown in the following formula (I-1), formula (I-2), and formula (I-3):

[0007]

[0008] R1 is selected from any one of the following structural formulae, wherein the wavy line represents a covalent bond to an adjacent structure:

[0009]

[0010] In one embodiment, R1 is

[0011] A mitochondrially targeted fluorinated ionizable lipid or a pharmaceutically acceptable salt, stereoisomer, or tautomer thereof, selected from the following compounds:

[0012]

[0013]

[0014] In another aspect, the present invention provides a mitochondrial-targeted lipid carrier, which comprises the fluorinated ionizable lipids, cationic lipids, auxiliary lipids, structural lipids, and functional polymer-conjugated lipids modified with mitochondrial targeting groups described in the present invention.

[0015] In one embodiment, the molar ratio of the fluorinated ionizable lipid to the carrier is 12.5% to 50%.

[0016] In one embodiment, the cationic lipid is selected from one or more of the following: trimethyl-2,3-dioleyloxypropylammonium chloride (DOTMA), trimethyl-2,3-dioleyloxypropylammonium bromide (DOTAP), O-[(N,N-dimethylaminoethyl)-carbamoyl]cholesterol hydrochloride (DC-Chol), 4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester (DLin-MC3-DMA), heptadecan-9-yl-8-((2-hydroxyethyl)(6-oxo-6-((decyloxy)hexyl)amino)octanoate) (SM-102), ((4-hydroxybutyl)azadialkyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315). In one embodiment, the cationic lipid is ALC-0315, and the molar ratio of the cationic lipid to the carrier is 12.5% to 50%.

[0017] In one embodiment, the helper lipid is selected from one or more of the following: dioleoylphosphatidylethanolamine (DOPE), dioleoylphosphatidylcholine (DOPC), distearoylphosphatidylcholine (DSPC), phosphatidic acid (PA), phosphatidylserine (PS), sphingomyelin (SM). In one embodiment, the helper lipid is DSPC.

[0018] In one embodiment, the structured lipid is selected from one or more of the following: cholesterol, non-sterols, sitosterol, ergosterol, α-tocopherol, corticosteroids. In one embodiment, the structured lipid is cholesterol.

[0019] In one embodiment, the carrier further comprises a polymer conjugated lipid containing a mitochondrial targeting group. The polymer conjugated lipid is selected from one or more of the following: distearoylphosphatidylethanolamine polyethylene glycol 2000-maleimide (DSPE-PEG 2000 -MAL), distearoylphosphatidylethanolamine polyethylene glycol 2000 (DSPE-PEG 2000 ), dimyristoylglycerol-3-methoxypolyethylene glycol 2000 (DMG-PEG 2000 ) and methoxypolyethylene glycol ditetradecyl acetamide (ALC-0159); the mitochondrial targeting group is selected from the mitochondrial targeting sequence (MTS) MLSLRQSIRFFKC, MLRAALSTARRGPRLSRLLC, and MVSGSSGLAAARL LSRTFLLQQNGIRHGSYC. In one embodiment, the MTS is MLSLRQSIRFFK C, which is grafted onto the carrier surface via a terminal thiol group reacting with a maleimide group of a polymer-conjugated lipid; the molar ratio of the polymer-conjugated lipid containing the mitochondrial targeting group to the carrier is 0.5% to 1.5%.

[0020] As a preferred embodiment of the present invention, in the lipid carrier, the molar ratio of the fluorinated ionizable lipid, cationic lipid, auxiliary lipid, structural lipid, and functional polymer conjugated lipid modified with a mitochondrial targeting group is (10-50): (10-40): (5-25): (10-55): (0.3-5).

[0021] In another aspect, the present invention provides a composition for mitochondrial gene delivery, comprising the fluorinated ionizable lipid compound of the present invention or a pharmaceutically acceptable salt, stereoisomer, tautomer thereof, or the lipid carrier of the present invention, and a functional gene.

[0022] In one embodiment, the functional gene in the mitochondrial gene delivery composition is selected from one or more of the following: human NADH ubiquinone oxidoreductase subunit 4 (ND4), human NADH ubiquinone oxidoreductase subunit 6 (ND6), and human NADH ubiquinone oxidoreductase subunit 1 (ND1). In one embodiment, the functional gene is ND4.

[0023] In one embodiment, the functional gene is located in a plasmid DNA vector, which, in addition to the nucleotide sequence encoding the functional gene, also encodes a Flag epitope tag sequence and a luciferase sequence for detecting mitochondrial transfection efficiency.

[0024] In one embodiment, the mitochondrial gene delivery composition is a nanoparticle preparation having an average particle size of 50 nm to 500 nm, preferably 100 nm to 200 nm; and a polydispersity index (PDI) of less than 50%, preferably less than 30%.

[0025] Another aspect of the present invention provides the mitochondrial gene delivery composition for use in treating diseases caused by mitochondrial gene mutations.

[0026] For example, the disease is selected from one or more of the following: LHON, mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes syndrome (MELAS), myoclonic epilepsy and ragged red fibers (MERRF), preferably LHON.

[0027] Beneficial effects of the present invention:

[0028] The fluorinated ionizable lipid gene delivery vector with mitochondrial targeting provided by the present invention has excellent biocompatibility. The functional fluorinated ionizable lipid and MTS in the vector simultaneously enhance the cellular uptake and mitochondrial accumulation of gene drugs, improving the efficiency of mitochondrial gene transfection and achieving efficient delivery of gene drugs to the mitochondrial matrix. By loading functional genes, it can be used to treat diseases caused by various mitochondrial gene mutations, such as LHON. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a schematic diagram of cell survival at different concentrations of different fluorinated carriers in Example 8 of the present invention.

[0030] Figure 2 This is a schematic diagram of the in vitro cellular uptake of different fluorinated carriers in Example 9 of the present invention.

[0031] Figure 3 Schematic diagram of in vitro mitochondrial transfection of different fluorinated vectors in Example 10 of the present invention.

[0032] Figure 4 This is a schematic diagram of the in vitro mitochondrial uptake of different vectors in Example 11 of the present invention.

[0033] Figure 5 Graph showing the co-localization coefficients of different vectors and mitochondria in Example 11 of the present invention.

[0034] Figure 6 This is a schematic diagram of the in vitro expression of target proteins by different vectors in Example 12 of the present invention.

[0035] Figure 7 This is a schematic diagram of the expression of the target protein in vivo of the optimal mitochondrial-targeted fluorinated vector preparation in Example 13 of the present invention. DETAILED DESCRIPTION

[0036] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0037] Example 1: Synthesis of fluorinated ionizable lipid compound 1 (4F)

[0038] The synthetic route is as follows:

[0039]

[0040] Synthesis of compound 1a: Sodium dithionite (3.14 g, 17.8 mmol) was slowly added to a mixture of octafluoro-1,4-diiodobutane (4.10 g, 9.0 mmol), vinyl benzoate (1.25 mL, 9.0 mmol), 1-hexene (1.41 mL, 9.0 mmol), and NaHCO₃ (1.91 g, 18.0 mmol) in CH₃CN / H₂O (8:7, v / v, 18.4 mL). The reaction mixture was stirred at 0°C for 50 min. After completion of the reaction, the pH was adjusted to 5-7 with 3N HCl and stirring was continued at room temperature for 20 min. The organic phase was extracted with CH₂Cl₂ and dried over MgSO₄. The product was purified by silica gel column chromatography using C₆H₂ as the eluent. 14 / CH2Cl2 (95:5, v / v) to afford compound 1a (1.42 g, 22%) as a slightly yellow waxy solid.

[0041] Synthesis of compound 1b: Compound 1a (1.42 g, 2.0 mmol) and AIBN (34.0 mg, 0.20 mmol) were dissolved in diethyl ether (50 mL), tributyltin hydride (2.65 mL, 10.0 mmol) was added, and the reaction mixture was refluxed under a halogen lamp for 15 h. The solvent was removed under reduced pressure and the product was purified by silica gel column chromatography using C6H2O as the eluent. 14 / CH2Cl2 (90:10, v / v) to afford compound 1b (0.74 g, 80%) as a white waxy solid.

[0042] Synthesis of Compound 1c: Compound 1b (0.69 g, 1.5 mmol) was stirred in 1 M LiOH (4 mL) in methanol at room temperature for 3 h. After vacuum drying, the product was dissolved in CH2Cl2, washed with water and saline, and the organic phase was dried over MgSO4. Purification was performed by silica gel column chromatography using C6H 14 / CH2Cl2 (50:50, v / v) to afford compound 1c (0.40 g, 75%) as a white waxy solid.

[0043] Synthesis of Compound 1d: Compound 1c (0.39 g, 1.1 mmol) was dissolved in acetone (40 mL) and water (0.5 mL). Jones reagent was added dropwise at room temperature until a persistent reddish-brown solution was obtained. The reaction mixture was stirred for 1 h, followed by the addition of i-PrOH (2 mL) and continued stirring for 15 min. The solvent was removed under reduced pressure, water (5 mL) was added, and the mixture was extracted with ether. The organic phase was washed with water and then with saline, and finally dried over MgSO4. Filtering and vacuum drying afforded Compound 1d (0.37 g, 91%) as a white waxy solid.

[0044] Synthesis of Compound 1: Compound 1d (0.37 g, 1.0 mmol), 3-(dipropylamino)propane-1,2-diol (96 mg, 0.5 mmol), DMAP (13 mg, 0.1 mmol), and DIC (126.2 mg, 1.0 mmol) were dissolved in CH2Cl2 (150 mL). After stirring at room temperature for 24 h, the precipitate was removed by filtration, and the filtrate was dried by rotary evaporation. Purification by silica gel column chromatography with petroleum ether / ethyl acetate (3:1, v / v) as eluent gave Compound 1 (0.31 g, 71%). 1 H NMR (600MHz, Chloroform-d): δ 5.22 (m, 1H), 4.48 (m, 1H), 4.29 (m, 1H), 3.14 (m, 2H), 2.60 (m, 2H), 2.39 (m, 4H), 2.03 (m, 4H), 1.31 (m, 30H), 0.86 (m, 12H). 19 F NMR (565MHz, Chloroform-d): δ-113.8 (m, 4F), -114.5 (m, 8F), -123.9 (m, 4F).

[0045] Example 2: Synthesis of fluorinated ionizable lipid compound 2 (6F)

[0046] The synthetic route is as follows:

[0047]

[0048] Synthesis of compound 2a: Sodium dithionite (3.14 g, 17.8 mmol) was slowly added to a mixture of dodecafluoro-1,6-diiodohexane (5.00 g, 9.0 mmol), vinyl benzoate (1.25 mL, 9.0 mmol), 1-hexene (1.12 mL, 9.0 mmol), and NaHCO₃ (1.91 g, 18.0 mmol) in CH₃CN / H₂O (8:7, v / v, 18.4 mL). The reaction mixture was stirred at 0°C for 50 min. After completion of the reaction, the pH was adjusted to 5-7 with 3N HCl and stirring was continued at room temperature for 20 min. The organic phase was extracted with CH₂Cl₂ and dried over MgSO₄. The product was purified by silica gel column chromatography using C₆H₂ as the eluent. 14 / CH2Cl2 (95:5, v / v) to afford compound 2a (1.51 g, 20%) as a slightly yellow waxy solid.

[0049] Synthesis of compound 2b: Compound 2a (1.5 g, 1.9 mmol) and AIBN (31 mg, 0.19 mmol) were dissolved in diethyl ether (50 mL), tributyltin hydride (2.56 mL, 9.5 mmol) was added, and the reaction mixture was refluxed under a halogen lamp for 15 h. The solvent was removed under reduced pressure and the product was purified by silica gel column chromatography using C6H2O as the eluent. 14 / CH2Cl2 (90:10, v / v) to afford compound 2b (0.84 g, 83%) as a white waxy solid.

[0050] Synthesis of Compound 2c: Compound 2b (0.80 g, 1.5 mmol) was stirred in 1 M LiOH (4 mL) in methanol at room temperature for 3 h. After vacuum drying, the product was dissolved in CH2Cl2, washed with water and saline, and the organic phase was dried over MgSO4. Purification was performed by silica gel column chromatography using C6H 14 / CH2Cl2 (50:50, v / v) to afford compound 2c (0.52 g, 80%) as a white waxy solid.

[0051] Synthesis of Compound 2d: Dissolve compound 2c (0.52 g, 1.2 mmol) in acetone (40 mL) and water (0.5 mL). Add Jones reagent dropwise at room temperature until a persistent reddish-brown solution is obtained. Stir the reaction mixture for 1 h, then add i-PrOH (2 mL) and continue stirring for 15 min. Remove the solvent under reduced pressure, add water (5 mL), and extract with ether. Wash the organic phase with water and saline, then dry over MgSO4. Filter and dry in vacuo to afford compound 2d (0.45 g, 85%) as a white waxy solid.

[0052] Synthesis of Compound 2: Compound 2d (0.44 g, 1.0 mmol), 3-(dipropylamino)propane-1,2-diol (70 mg, 0.4 mmol), DMAP (10 mg, 0.08 mmol), and DIC (2.9 g, 23 mmol) were dissolved in CHCl (150 mL). After stirring at room temperature for 24 h, the precipitate was removed by filtration, and the filtrate was dried by rotary evaporation. Purification by silica gel column chromatography with petroleum ether / ethyl acetate (3:1, v / v) as eluent gave Compound 2 (0.36 g, 35%). 1 H NMR (600MHz, Chloroform-d): δ 5.22 (m, 1H), 4.56 (m, 1H), 4.31 (m, 1H), 3.15 (m, 2H), 2.61 (m, 2H), 2.40 (m, 4H), 2.03 (m, 4H), 1.40 (m, 22H), 0.87 (m, 12H). 19 F NMR (565MHz, Chloroform-d): δ -111.6 (m, 4F), -114.5 (m, 4F), -118.8 (m, 8F), -121.9 (m, 4F), -123.7 (m, 4F).

[0053] Example 3: Synthesis of fluorinated ionizable lipid compound 3 (8F)

[0054] The synthetic route is as follows:

[0055]

[0056] Synthesis of compound 3a: Sodium dithionite (1.76 g, 10.0 mmol) was slowly added to a mixture of hexadecafluoro-1,8-diiodooctane (3.3 g, 5.0 mmol), vinyl benzoate (0.7 mL, 5.0 mmol), 1-bromobutene (0.68 g, 5.0 mmol), and NaHCO₃ (0.84 g, 10.0 mmol) in CH₃CN / H₂O (8:7, v / v, 18.4 mL). The reaction mixture was stirred at 0°C for 50 min. After completion of the reaction, the pH was adjusted to 5-7 with 3N HCl and stirring was continued at room temperature for 20 min. The organic phase was extracted with CH₂Cl₂ and dried over MgSO₄. The product was purified by silica gel column chromatography using C₆H₂ as the eluent. 14 / CH2Cl2 (95:5, v / v) to afford compound 3a (0.94 g, 20%) as a slightly yellow waxy solid.

[0057] Synthesis of compound 3b: Compound 3a (0.94 g, 1.0 mmol) and AIBN (34.0 mg, 0.20 mmol) were dissolved in toluene (50 mL), tributyltin hydride (2.65 mL, 10.0 mmol) was added, and the reaction mixture was refluxed under a halogen lamp for 15 h. The solvent was removed under reduced pressure and the product was purified by silica gel column chromatography using C6H2O as the eluent. 14 / CH2Cl2 (90:10, v / v) to afford compound 3b (0.32 g, 53%) as a white waxy solid.

[0058] Synthesis of compound 3c: Compound 3b (0.32 g, 0.53 mmol) was stirred in 1 M LiOH (3 mL) in methanol at room temperature for 3 h. After vacuum drying, the product was dissolved in CH2Cl2, washed with water and saline, and the organic phase was dried over MgSO4. Purification was performed by silica gel column chromatography using C6H 14 / CH2Cl2 (50:50, v / v) to afford compound 3c (0.19 g, 72%) as a white waxy solid.

[0059] Synthesis of Compound 3d: Compound 3c (0.19 g, 0.38 mmol) was dissolved in acetone (20 mL) and water (0.2 mL). Jones reagent was added dropwise at room temperature until a persistent reddish-brown solution was obtained. The reaction mixture was stirred for 1 h, followed by the addition of i-PrOH (2 mL) and continued stirring for 15 min. The solvent was removed under reduced pressure, water (5 mL) was added, and the mixture was extracted with ether. The organic phase was washed with water and saline, then dried over MgSO4. Filtering and vacuum drying afforded compound 3d (0.16 g, 81%) as a white waxy solid.

[0060] Synthesis of Compound 3: Compound 3d (0.16 g, 0.3 mmol), 3-(dipropylamino)propane-1,2-diol (18 mg, 0.1 mmol), DMAP (13 mg, 0.1 mmol), and DIC (38.2 mg, 0.3 mmol) were dissolved in CH2Cl2 (50 mL). After stirring at room temperature for 24 h, the precipitate was removed by filtration, and the filtrate was dried by rotary evaporation. Purification by silica gel column chromatography with petroleum ether / ethyl acetate (3:1, v / v) as eluent gave Compound 3 (65.6 mg, 56%). 1 H NMR (600MHz, Chloroform-d): δ 5.17 (m, 1H), 4.54 (m, 1H), 4.23 (m, 1H), 3.15 (m, 2H), 2.67 (t, 4H), 2.56 (t, 2H), 2.38 (m, 4H), 1.38 (m, 14H), 0.87 (m, 12H). 19F NMR (565MHz, Chloroform-d): δ -111.8 (m, 4F), -114.1 (m, 4F), -114.5 (m, 16F), -123.5 (m, 4F), -124.8 (m, 4F).

[0061] Example 4: Synthesis of non-fluorinated ionizable lipid compounds (OF)

[0062] The synthetic route is as follows:

[0063]

[0064] Synthesis of Compound 4: Compound 4a (0.45 g, 0.3 mmol), 3-(dipropylamino)propane-1,2-diol (54 mg, 0.3 mmol), DMAP (38 mg, 0.3 mmol), and DIC (95.5 mg, 0.75 mmol) were dissolved in CH2Cl2 (50 mL). After stirring at room temperature for 24 h, the precipitate was removed by filtration, and the filtrate was dried by rotary evaporation. Purification by silica gel column chromatography with petroleum ether / ethyl acetate (5:1, v / v) as eluent gave Compound 4 (0.13 g, 70%). 1 H NMR (600MHz, Chloroform-d): δ5.10 (m, 1H), 4.35 (dd, 1H), 4.12 (dd, 1H), 2.52 (m, 2H ), 2.37(m, 4H), 2.28(m, 4H), 1.59(m, 4H), 1.40(m, 4H), 1.26(m, 40H), 0.86(m, 12H).

[0065] Example 5: Preparation of mitochondrial-targeted fluorinated vector formulations

[0066] The fluorinated ionizable lipid compounds 1 to 3 (4F, 6F, 8F) in Examples 1 to 3 were respectively mixed with the representative cationic lipids ALC-0315, DSPC, cholesterol, DSPE-PEG 2000Dissolve in the first solution at the molar ratio in Table 1 to prepare a solution with a total lipid concentration of 1.28 mg / mL for standby use. Take the plasmid DNA solution and dilute it in the second solution to prepare a diluent with a plasmid DNA concentration of 0.013 mg / mL for standby use. Wherein, the first solution is an ethanol solution, and the second solution is a 10mM citric acid buffered saline solution with a pH of 4.0, with a volume ratio of 1:3. The two phases are quickly mixed using a microfluidic chip, and the ethanol in the system is removed by ultrafiltration and replaced with a NaCl solution to obtain a series of gene-carrying fluorinated preparations 4F-LNP@DNA, 6F-LNP@DNA, and 8F-LNP@DNA. At the same time, the gene-carrying non-fluorinated preparation OF-LNP@DNA prepared by the same method using the non-fluorinated ionizable lipid (OF) in Example 4 and the gene-carrying non-fluorinated preparation LNP@DNA prepared by the same method using the ALC-0315 cationic lipid alone are used as controls. The percentages in the groups in Table 1 represent the molar percentage of fluorinated ionizable lipids to total cationic lipids.

[0067] Table 1 Molar ratio of fluorinated / non-fluorinated ionizable lipids and other lipids in the first solution

[0068]

[0069]

[0070] Example 6: Preparation of Optimal Fluorinated Vector Formulation for Mitochondrial Targeting

[0071] The fluorinated ionizable lipid compound 2 (6F) in Example 2 was mixed with ALC-0315, DSPC, cholesterol, DSPE-PEG 2000 , DSPE-PEG 2000 -MAL was dissolved in the first solution at the molar ratios shown in Table 2 to prepare a solution with a total lipid concentration of 1.28 mg / mL, which was set aside. The plasmid DNA solution was diluted in the second solution to prepare a dilution with a plasmid DNA concentration of 0.013 mg / mL, which was set aside. The first solution was an ethanol solution, and the second solution was a 10 mM citric acid buffered saline solution at pH 4.0, with a volume ratio of 1:3. The two phases were rapidly mixed using a microfluidic chip, and the ethanol in the system was removed by ultrafiltration and replaced with a NaCl solution. An appropriate amount of MTS solution was added to the solution and allowed to react overnight at 4°C. The MTS terminal thiol group reacted with the maleimide group on the outer side of the carrier to obtain the gene-carrying MTS-containing fluorinated formulation 6F-M-LNP@DNA. At the same time, the non-fluorinated ionizable lipid (OF) described in Example 4 was used to prepare the gene-carrying MTS-containing non-fluorinated formulation OF-M-LNP@DNA in the same manner as a control.

[0072] Table 2 Molar ratio of fluorinated / non-fluorinated ionizable lipids and other lipids in the first solution

[0073]

[0074] Example 7: Physicochemical Characterization of Different Carrier Formulations

[0075] The particle size, polydispersity index (PDI), and zeta potential of the carrier@DNA formulations prepared in Examples 5 and 6 were measured using a Malvern laser particle size analyzer. 40 μL of each carrier@DNA solution was diluted to 1 mL with deionized water and added to the sample cell. Each sample was measured three times. The results are shown in Table 3. The lipid nanoparticles prepared in Examples 5 and 6 all had particle sizes between 100 nm and 200 nm, PDIs between 0.1 and 0.4, and zeta potentials between -20 and 0 mV.

[0076] Table 3 Particle size, PDI and potential characterization of each carrier preparation@DNA

[0077]

[0078]

[0079] Example 8: In vitro cytotoxicity of different fluorinated carriers

[0080] L02 cells were seeded in 96-well plates (1×10 4 Cells were cultured overnight in a cell culture incubator and treated with different fluorinated carriers (0F, 4F, 6F, 8F) in Example 5. The cells were then incubated in the incubator for 72 hours. 20 μL of thiazolyl blue (MTT) (5 mg / mL) solution was then added and incubated for 4 hours. The supernatant was aspirated and 150 μL of DMSO was added to dissolve the precipitate in the 96-well plate. The ultraviolet absorption was measured at 490 nm. The cell survival rate under different fluorinated carriers and different fluorinated lipid concentrations was calculated. The results are shown in FIG. Figure 1 The results of the MTT assay showed that the cell viability of different fluorinated carriers remained above 90% within the fluorinated lipid concentration range of 10-60 μg / mL, demonstrating good in vitro safety.

[0081] Example 9: In vitro cellular uptake of different fluorinated carriers

[0082] L02 cells were seeded in 24-well plates (8×10 4Cells were cultured overnight in a cell culture incubator. The cells were treated with different carriers@Cy5-DNA complex nanoparticles (containing 1 μg Cy5-DNA) in Example 5 and incubated in the incubator for 24 hours. The supernatant was aspirated, washed once with PBS, 0.2 mL of trypsin was added to digest the cells, and the digested cells were collected into a centrifuge tube using fresh culture medium. Finally, 0.2 mL of PBS was added to resuspend the cells and transferred to a flow cytometer for use. The Cy5 fluorescence intensity was quantitatively detected on a flow cytometer to investigate the cellular uptake of nanoparticles. The results are shown in Figure 2. Figure 2 As shown in the data, the fluorinated preparation group generally showed a higher cellular uptake level than the non-fluorinated preparation group, and the fluorescence intensity differences between different fluorinated carrier groups with different fluorinated ionizable lipid concentrations were obvious, among which 50%-6F-LNP and 25%-8F-LNP had the best cellular uptake effect.

[0083] Example 10: In vitro mitochondrial transfection with different fluorinated vectors

[0084] L02 cells were seeded in 6-well plates (2×10 5 cells / well), incubated overnight in a cell culture incubator, treated with different vector@DNA complex nanoparticles (containing 2μg DNA) in Example 5, changed the medium after incubation for 24h, and continued to culture for 48h. Aspirate the supernatant, wash once with PBS, add 0.3mL of trypsin to digest the cells, and use fresh culture medium to collect the digested cells into a centrifuge tube. After the cells were fixed and permeabilized, add Flag primary antibody and incubate at room temperature for 30min. Subsequently, aspirate the supernatant, wash once with PBS, add the corresponding Alexa Fluor 647 fluorescent secondary antibody, and continue to incubate at room temperature for 30min. Finally, aspirate the supernatant, wash once with PBS, add 0.2mL PBS to resuspend, transfer to a flow tube for use, and quantitatively detect the Alexa Fluor 647 fluorescence intensity on a flow cytometer to investigate the in vitro mitochondrial transfection of different preparation groups. The results are as follows Figure 3As shown in Table 4, the fluorescence intensity of the non-fluorinated preparation group was lower, and the fluorescence intensity of the different fluorinated carrier groups with different fluorinated ionizable lipid concentrations was significantly different, indicating that the different fluorinated carrier groups had different mitochondrial transfection abilities, among which 50%-6F-LNP had the best mitochondrial transfection ability. In addition, the transfection abilities of different carrier@DNA complex nanoparticles (50%-OF-LNP, 50%-4F-LNP, 50%-6F-LNP, and 50%-8F-LNP) prepared with the same fluorinated / non-fluorinated ionizable lipid molar ratio were compared. The results are shown in Table 4. The fluorescence intensity of the 50%-6F-LNP group was 2.37 times that of the 50%-OF-LNP group, 2.21 times that of the 50%-4F-LNP group, and 1.42 times that of the 50%-8F-LNP group. The one-way analysis of variance (ANOVA) showed that the 50%-6F-LNP group had highly significant differences compared with the other groups (p < 0.001), and the mitochondrial transfection ability was significantly improved.

[0085] Table 4 Fluorescence intensity detection results after transfection of different vector@DNA complex nanoparticles

[0086] Group Mean fluorescence intensity (MFI) 50%-0F-LNP 76.5±1.5 50%-4F-LNP 82.1±1.7 50%-6F-LNP 181.3±1.2 50%-8F-LNP 127.7±1.5

[0087] Example 11: Mitochondrial uptake of different vectors in vitro

[0088] L02 cells were seeded in 35 mm glass culture dishes (5 × 10 4 Cells / dish), incubated overnight in a cell culture incubator, treated with different carriers LNP, OF-LNP (i.e. 50%-OF-LNP), OF-M-LNP, 6F-LNP (i.e. 50%-6F-LNP), 6F-M-LNP@Cy5-DNA complex nanoparticles (containing 1 μg DNA) and incubated for 24 hours. Among them, the LNP group prepared separately by the representative cationic lipid ALC-0315 in the prior art was used as the positive control group. Subsequently, the supernatant was aspirated, washed once with PBS, and 1 mL of mitochondrial green fluorescent probe was added to stain at 37°C for 30 minutes. After washing with PBS, it was observed under a laser confocal microscope, and the CLSM images were analyzed for mitochondrial co-localization using ImageJ software. Mitochondria exhibited green fluorescence after being stained with green fluorescent dye, Cy5-DNA exhibited red fluorescence, and yellow indicated the co-localization of the carrier and mitochondria. The results are shown in the figure below. Figure 4 As shown in Figure 2, the fluorinated preparation group 6F-LNP and 6F-M-LNP showed stronger yellow fluorescence than the other groups. ANOVA test was used to compare the colocalization coefficient means among multiple groups. The results are shown in Figure 2. Figure 5As shown in the figure, ns indicates p>0.05, not significantly different; * indicates p<0.05, significantly different; ** indicates p<0.01, moderately significantly different; *** indicates p<0.001, highly significantly different. It can be seen that the optimal formulation, 6F-M-LNP, has the highest mitochondrial colocalization coefficient, indicating that it has the best mitochondrial targeting efficiency.

[0089] Example 12: In vitro expression of target proteins using different vectors

[0090] L02 cells were seeded in a 10 cm cell culture dish (1.5×10 6 Cells / dish), incubated overnight in a cell culture incubator, treated with different carriers LNP, OF-LNP, OF-M-LNP, 6F-LNP, 6F-M-LNP@DNA complex nanoparticles (containing 10μgDNA), incubated for 24h, and the solution was changed and cultured for another 48h. The cells treated with each preparation group were collected, mitochondrial proteins were extracted, and samples were loaded for SDS-PAGE gel electrophoresis and Western Blot. The ND4 target protein primary antibody and the mitochondrial internal reference COX IV protein primary antibody and the corresponding secondary antibody were incubated and developed. The protein bands were quantitatively analyzed by ImageJ software to investigate the expression of mitochondrial ND4 protein in each preparation group. Among them, the LNP group prepared by the representative cationic lipid ALC-0315 alone in the prior art was used as the positive control group. The ANOVA test was used to compare the means among multiple groups, and the results are as follows. Figure 6 As shown in the figure, * indicates p < 0.05, which is a significant difference; *** indicates p < 0.001, which is a highly significant difference. It can be seen that the ND4 protein expression levels of the fluorinated preparation groups 6F-LNP and 6F-M-LNP are significantly different from those of the non-fluorinated preparation group, and the ND4 protein expression level of the 6F-M-LNP group is the highest, indicating that the optimal preparation 6F-M-LNP group can achieve higher mitochondrial gene transfection and target protein expression.

[0091] Example 13: In vivo expression of target protein using the optimal formulation of mitochondrial-targeted fluorinated vector

[0092] The optimal formulation 6F-M-LNP@DNA complex nanoparticles in Example 6 were injected into the mouse eyes (1 μg DNA / eye) by intravitreal administration. After 3, 7, 14, and 30 days of administration, the mouse eyeballs were collected, mitochondrial proteins were extracted, and the samples were loaded for SDS-PAGE gel electrophoresis and Western Blot. The ND4 target protein primary antibody and the mitochondrial internal reference COX IV protein primary antibody and the corresponding secondary antibody were incubated and developed. The protein bands were quantitatively analyzed by ImageJ software to investigate the protein expression level and duration of the ND4 gene delivered by the optimal formulation FM-LNP in mice. The ANOVA test was used to compare the means among multiple groups. The results are shown in Figure 2. Figure 7 As shown in the figure, ns indicates p>0.05, indicating no significant difference; *** indicates p<0.001, indicating a highly significant difference. It can be seen that the 6F-M-LNP delivery vector can effectively deliver the ND4 gene into the mouse eyeball, achieving mitochondrial gene transfection and sustained expression for 30 days.

[0093] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A mitochondrial-targeted fluorinated ionizable lipid or a pharmaceutically acceptable salt, stereoisomer, or tautomer thereof, characterized in that The structure of the fluorinated ionizable lipid is shown in formula (I): Wherein, a=an integer of 2 to 8, b=an integer of 4 to 8; R1 is= 2. The compound of formula (I) or a pharmaceutically acceptable salt, stereoisomer, or tautomer thereof according to claim 1, characterized in that: The compound of formula (I) has one of the following structures:

3. A mitochondrial-targeted lipid carrier, characterized in that: The carrier comprises the fluorinated ionizable lipid, cationic lipid, auxiliary lipid, structural lipid, or functional polymer conjugated lipid modified with a mitochondrial targeting group according to any one of claims 1 to 2.

4. The lipid carrier according to claim 3, characterized in that The cationic lipid is selected from one or more of the following: DOTMA, DOTAP, DC-Chol, DLin-MC3-DMA, SM-102, ALC-0315; the auxiliary lipid is selected from one or more of the following: DOPE, DOPC, DSPC, PA, PS, SM; the structural lipid is selected from one or more of the following: cholesterol, non-sterol, sitosterol, ergosterol, α-tocopherol, corticosteroid; the polymer conjugated lipid is selected from one or more of the following: DSPE-PEG 2000 -MAL, DSPE-PEG 2000 , DMG-PEG 2000 , ALC-0159; the mitochondrial targeting group is selected from any one of the mitochondrial targeting sequences: MLSLRQSIRFFKC, MLRAALSTARRGPRLSRLLC, MVSGSSGLAAARLLSRTFLLQQNGIRHGSYC; in the lipid carrier, the molar ratio of the fluorinated ionizable lipid, cationic lipid, auxiliary lipid, structural lipid, and functional polymer conjugated lipid modified with a mitochondrial targeting group is (10-50): (10-40): (5-25): (10-55): (0.3-5).

5. The lipid carrier according to claim 4, characterized in that The cationic lipid is selected from one or more of the following: ALC-0315.

6. The lipid carrier according to claim 4, characterized in that The helper lipid is selected from DSPC.

7. The lipid carrier according to claim 4, characterized in that The structural lipid is selected from cholesterol.

8. The lipid carrier according to claim 3, characterized in that The polymer conjugated lipid is selected from DSPE-PEG 2000 -MAL and / or DSPE-PEG 2000 .

9. The lipid carrier according to claim 3, characterized in that The mitochondrial targeting group is selected from MLSLRQSIRFFKC.

10. A composition for mitochondrial gene delivery, characterized in that The composition comprises the fluorinated ionizable lipid compound according to any one of claims 1 to 2 or a pharmaceutically acceptable salt, stereoisomer, or tautomer thereof, or the lipid carrier according to any one of claims 3 to 9, and a functional gene.

11. The composition according to claim 10, characterized in that The composition is a nanoparticle preparation, the average particle size of the nanoparticle preparation is between 50nm and 500nm, and the polydispersity index (PDI) of the nanoparticle preparation is less than 50%.

12. The composition according to claim 10, characterized in that The functional gene is a plasmid DNA vector containing the target gene.

13. Use of the fluorinated ionizable lipid compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 2, or the lipid carrier according to any one of claims 3 to 9, or the composition according to claim 10 in the preparation of a medicament for treating a disease caused by a mitochondrial gene mutation.

14. The use according to claim 13, characterized in that The disease is Leber hereditary optic neuropathy.

15. The use according to claim 14, characterized in that The functional gene in the composition is selected from one of human NADH ubiquinone oxidoreductase subunit 4, human NADH ubiquinone oxidoreductase subunit 6 and human NADH ubiquinone oxidoreductase subunit 1.

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