Triglyceride-like ionizable lipid as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of drug carriers, and relates to a triglyceride-like ionizable lipid as well as a preparation method and application thereof, in particular to a triglyceride-like ionizable lipid as shown in the following formula as well as a preparation method and application thereof. On the basis of the structure of triglyceride, two fatty acid tail chains and a tertiary amino head can be introduced to form a general structure conforming to ionizable lipid. The triglyceride skeleton improves the degradability of the lipid, and the lysosome escape ability of the lipid nanoparticles is improved by adjusting the position of the tertiary amine head group and the length and saturation of the fatty acid tail chain. Experiments show that the 2-TG-LA-LNPs prepared by taking the 2-TG-LA as the ionizable lipid is superior to Dlin-MC3-DMA in the aspects of degradability, lysosome escape and liver delivery of siRNA, and a new strategy is expected to be provided for nucleic acid delivery. # imgabs0 #

Description

Technical Field

[0001] The invention belongs to the technical field of drug carriers, and in particular relates to ionizable lipids based on triglyceride structures, and a preparation method and application thereof. Background Art

[0002] Lipid nanoparticles are considered to be one of the most promising platforms for nucleic acid drug delivery. However, most lipid nanoparticles still face the problems of poor biodegradability and low lysosomal escape efficiency in vivo. The introduction of ester bonds in the lipid structure can improve the degradability of lipid materials. Triglycerides are glycerides generated by esterification of three hydroxyl groups of glycerol with three fatty acid molecules. Because they contain multiple ester bonds, they show excellent degradability. Based on the structure of triglycerides, two fatty acid tail chains and a tertiary amine head can be introduced to form a general structure that conforms to ionizable lipids. The head and tail structures are important components of ionizable materials, affecting the lysosomal escape ability and transfection activity of lipid materials.

[0003] Based on the above, the inventors combined the degradable triglyceride structure with the head of different chain lengths and the fatty acid tail chains of different chain lengths and saturations to design triglyceride-like ionizable lipids, which not only improved the degradability of the lipid material, but also improved the lysosomal escape ability of the lipid nanoparticles. Summary of the invention

[0004] The present invention overcomes the defects and shortcomings in the prior art and provides a novel triglyceride-like ionizable lipid and a preparation method and application thereof.

[0005] In order to achieve the purpose of the present invention, the present invention adopts the following technical scheme:

[0006] The first object of the present invention is to provide a triglyceride-like ionizable lipid. The triglyceride-like ionizable lipid of the present invention has the following structural formula:

[0007]

[0008] wherein n is 2, 3 or 4, R1 and R2 are each independently selected from -(CH2)7CH=CHCH2CH=CH(CH2)4CH3, -(CH2)7CH=CH(CH2)7CH3, -(CH2) 12 CH3, -(CH2) 14 CH3 or -(CH2) 16 CH3.

[0009] As an optional method, in the above-mentioned triglyceride-like ionizable lipids, the tertiary amine head group is N,N-dimethyl tertiary amine, and is located at the β-, γ- or δ-position of the ester bond, respectively; the connecting region is triglyceride; and the fatty acid tail chain is myristic acid, palmitic acid, stearic acid, oleic acid or linoleic acid.

[0010] As an optional mode, in the above-mentioned triglyceride-like ionizable lipid, the triglyceride-like ionizable lipid is selected from the following compounds:

[0011]

[0012] The second object of the present invention is to provide a method for preparing the triglyceride-like ionizable lipids described in the first object.

[0013] The present invention adopts the following method to synthesize the above-mentioned triglyceride-like ionizable lipid, and the synthesis route is as follows:

[0014] The synthesis method of the triglyceride-like ionizable lipid is to connect 1,3-dihydroxyacetone with fatty acid through an esterification reaction under the action of a catalyst and obtain a triglyceride-like tail structure through reduction, and connect the tail structure with an acid containing a tertiary amino head through an esterification reaction to obtain the triglyceride-like ionizable lipid;

[0015]

[0016] The catalysts are 4-dimethylaminopyridine (DMAP) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI).

[0017] The third object of the present invention is to provide the use of the triglyceride-like ionizable lipid described in the first object in the preparation of nucleic acid drug delivery vectors.

[0018] As an alternative, in the above application, the delivery vector is used for the delivery of one or more of pDNA, siRNA, ASO or mRNA.

[0019] As an alternative, in the above application, the delivery vehicle is a lipid nanoparticle.

[0020] As an optional method, in the above application, the nucleic acid drug delivery vector is locally administered through muscle, subcutaneous, intradermal, intratumoral microneedle, injection or perfusion, or the nucleic acid drug delivery vector is administered by intravenous injection.

[0021] The degradability of ionizable lipids is an important factor affecting lipid applications, so investigating the release of ionizable lipids is conducive to predicting the degradability of lipids in vivo. siRNA quickly and effectively escapes from lysosomes into the cytoplasm to form RNA-induced silencing complexes, which is of great significance to gene silencing, so investigating the lysosomal escape efficiency of lipid nanoparticles is conducive to predicting the gene silencing efficiency of lipid nanoparticles in vivo. The present invention is expected to provide lipids that can significantly improve the degradability of ionizable lipids and the lysosomal escape efficiency, for nucleic acid drug delivery.

[0022] A list of some triglyceride-like ionizable lipids synthesized by the present invention is shown in Table 1.

[0023] Table 1 Structures and structural identification information of triglyceride-like ionizable lipids

[0024]

[0025]

[0026] Beneficial effects of the present invention:

[0027] The invention introduces a triglyceride backbone to improve the degradability of ionizable lipids, introduces heads with different chain lengths and fatty acid tail chains with different chain lengths and saturations, and screens out ionizable lipids with higher lysosomal escape ability for nucleic acid drug delivery, which is of great significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Release of ionizable lipids containing triglycerides of different head groups.

[0029] Figure 2 Pearson colocalization coefficients of triglyceride-like ionizable lipids containing different head groups.

[0030] Figure 3 In vivo gene silencing effects of triglyceride-like ionizable lipids containing different head groups. DETAILED DESCRIPTION

[0031] The present invention will be further described with reference to specific embodiments below. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the scope of the present invention.

[0032] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0033] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available products unless otherwise specified.

[0034] According to the general synthetic route of triglyceride-like ionizable lipids, the following compounds were prepared respectively.

[0035] Example 1

[0036] Linoleic acid (35.00mmol), DMAP (17.80mmol), and EDCI (35.70mmol) were added to an eggplant-shaped bottle and dissolved with anhydrous dichloromethane. Activated for 2h under ice bath, 1,3-dihydroxyacetone (15.90mmol) was added to the above reaction solution and reacted at room temperature for 12h. The above reactions were all carried out under N2 protection. After separation and purification by silica gel column chromatography, a colorless oily product was obtained.

[0037] The colorless oily product (7.65 mmol) was dissolved in tetrahydrofuran: benzene (40 mL: 8 mL), and 7 mL of distilled water was added under stirring. Sodium borohydride (29.87 mmol) was added under ice bath, and 3.39 mL of glacial acetic acid was added to terminate the reaction after stirring for 0.5 h, and 40 mL of anhydrous chloroform was added to the reaction solution. The reaction solution was washed twice with 5% sodium bicarbonate solution and once with saturated sodium chloride solution, and the organic layer was separated by extraction. An appropriate amount of anhydrous sodium sulfate was added to the organic layer to remove moisture, and the anhydrous sodium sulfate was removed by suction filtration. After removing the organic solvent with a rotary evaporator, a colorless oily product was obtained.

[0038] 3-Dimethylaminopropionic acid hydrochloride (3.20mmol), DMAP (1.80mmol), EDCI (4.00mmol) were added to a 100mL eggplant-shaped bottle and dissolved with 60mL of anhydrous chloroform. Activated for 2h under ice bath, and the colorless oily product (1.60mmol) was added to the reaction solution and reacted at room temperature for 12h. The above reactions were all carried out under N2 protection. The compound 2-TG-LA was obtained by separation and purification by silica gel column chromatography.

[0039] Example 2

[0040] Linoleic acid (35.00mmol), DMAP (17.80mmol), and EDCI (35.70mmol) were added to an eggplant-shaped bottle and dissolved with anhydrous dichloromethane. Activated for 2h under ice bath, 1,3-dihydroxyacetone (15.90mmol) was added to the above reaction solution and reacted at room temperature for 12h. The above reactions were all carried out under N2 protection. After separation and purification by silica gel column chromatography, a colorless oily product was obtained.

[0041] The colorless oily product (7.65 mmol) was dissolved in tetrahydrofuran: benzene (40 mL: 8 mL), and 7 mL of distilled water was added under stirring. Sodium borohydride (29.87 mmol) was added under ice bath, and 3.39 mL of glacial acetic acid was added to terminate the reaction after stirring for 0.5 h, and 40 mL of anhydrous chloroform was added to the reaction solution. The reaction solution was washed twice with 5% sodium bicarbonate solution and once with saturated sodium chloride solution, and the organic layer was separated by extraction. An appropriate amount of anhydrous sodium sulfate was added to the organic layer to remove moisture, and anhydrous sodium sulfate was removed by suction filtration. After removing the organic solvent with a rotary evaporator, a colorless oily product was obtained.

[0042] 4-Dimethylaminobutyric acid hydrochloride (12.92mmol), DMAP (7.30mmol), EDCI (16.15mmol) were added to an eggplant-shaped bottle and dissolved with anhydrous chloroform. Activated for 2h under ice bath, and the colorless oily product (6.46mmol) was added to the reaction solution and reacted at room temperature for 12h. The above reactions were all carried out under N2 protection. Compound 3-TG-LA was obtained by separation and purification by silica gel column chromatography.

[0043] Example 3

[0044] Linoleic acid (35.00mmol), DMAP (17.80mmol), and EDCI (35.70mmol) were added to an eggplant-shaped bottle and dissolved with anhydrous dichloromethane. Activated for 2h under ice bath, 1,3-dihydroxyacetone (15.90mmol) was added to the above reaction solution and reacted at room temperature for 12h. The above reactions were all carried out under N2 protection. After separation and purification by silica gel column chromatography, a colorless oily product was obtained.

[0045] The colorless oily product (7.65 mmol) was dissolved in tetrahydrofuran: benzene (40 mL: 8 mL), and 7 mL of distilled water was added under stirring. Sodium borohydride (29.87 mmol) was added under ice bath, and 3.39 mL of glacial acetic acid was added to terminate the reaction after stirring for 0.5 h, and 40 mL of anhydrous chloroform was added to the reaction solution. The reaction solution was washed twice with 5% sodium bicarbonate solution and once with saturated sodium chloride solution, and the organic layer was separated by extraction. An appropriate amount of anhydrous sodium sulfate was added to the organic layer to remove moisture, and the anhydrous sodium sulfate was removed by suction filtration. After removing the organic solvent with a rotary evaporator, a colorless oily product was obtained.

[0046] 5-Dimethylaminovaleric acid hydrochloride (3.20mmol), DMAP (1.80mmol), EDCI (4.00mmol) were added to an eggplant-shaped bottle and dissolved with anhydrous chloroform. Activated for 2h under ice bath, and the colorless oily product (1.60mmol) was added to the reaction solution and reacted at room temperature for 12h. The above reactions were all carried out under N2 protection. The compound 4-TG-LA was obtained by separation and purification by silica gel column chromatography.

[0047] Example 4

[0048] Myristic acid (43.00mmol), DMAP (17.20mmol), and EDCI (54.00mmol) were added to an eggplant-shaped bottle and dissolved in anhydrous chloroform. Activated for 2h in an ice bath, 1,3-dihydroxyacetone (14.00mmol) was added to the above reaction solution, and reacted at room temperature for 12h. The above reactions were all carried out under N2 protection. The reaction solution was spin-dried, and 200mL of anhydrous dichloromethane was added, and it was placed in a -20℃ refrigerator and frozen for 4h. After crystallization, a white solid product was obtained by suction filtration.

[0049] The above white solid product (10.78mmol) was dissolved in tetrahydrofuran: benzene (70mL: 14mL), and 7mL of distilled water was added under stirring. Sodium borohydride (33.00mmol) was added under ice bath conditions, and after stirring for 0.5h, 3.75mL of glacial acetic acid was added to terminate the reaction, and 70mL of anhydrous chloroform was added to the reaction solution. The above reaction solution was washed twice with 5% sodium bicarbonate solution and once with saturated sodium chloride solution, and the organic layer was separated by extraction. An appropriate amount of anhydrous sodium sulfate was added to the organic layer to remove moisture, and anhydrous sodium sulfate was removed by suction filtration. After removing the organic solvent with a rotary evaporator, a white solid product was obtained.

[0050] 4-Dimethylaminobutyric acid hydrochloride (19.54 mmol), DMAP (11.04 mmol), and EDCI (24.43 mmol) were added to an eggplant-shaped bottle and dissolved in anhydrous chloroform. Activated for 2 h in an ice bath, and the above white solid product (9.77 mmol) was added to the above reaction solution and reacted at room temperature for 12 h. Silica gel column chromatography was used for separation and purification to obtain compound 3-TG-14.

[0051] Example 5

[0052] Palmitic acid (39.00mmol), DMAP (16.20mmol), and EDCI (49.00mmol) were added to an eggplant-shaped bottle and dissolved in anhydrous chloroform. Activated for 2h in an ice bath, 1,3-dihydroxyacetone (13.00mmol) was added to the above reaction solution, and reacted at room temperature for 12h. The above reactions were all carried out under N2 protection. The reaction solution was spin-dried, and 200mL of anhydrous dichloromethane was added, and it was placed in a -20℃ refrigerator and frozen for 4h. After crystallization, a white solid product was obtained by suction filtration.

[0053] The above white solid product (9.89mmol) was dissolved in tetrahydrofuran: benzene (70mL: 14mL), and 7mL of distilled water was added under stirring. Sodium borohydride (33.84mmol) was added under ice bath, and 3.84mL of glacial acetic acid was added to terminate the reaction after stirring for 0.5h, and 70mL of anhydrous chloroform was added to the reaction solution. The above reaction solution was washed twice with 5% sodium bicarbonate solution and once with saturated sodium chloride solution, and the organic layer was separated by extraction. An appropriate amount of anhydrous sodium sulfate was added to the organic layer to remove moisture, and the anhydrous sodium sulfate was removed by suction filtration. After removing the organic solvent with a rotary evaporator, a white solid product was obtained.

[0054] 4-Dimethylaminobutyric acid hydrochloride (14.08 mmol), DMAP (7.96 mmol), and EDCI (17.61 mmol) were added to an eggplant-shaped bottle and dissolved in anhydrous chloroform. Activated for 2 h in an ice bath, and the above white solid product (7.04 mmol) was added to the above reaction solution and reacted at room temperature for 12 h. Silica gel column chromatography was used for separation and purification to obtain compound 3-TG-16.

[0055] Example 6

[0056] Stearic acid (35.00mmol), DMAP (14.00mmol), and EDCI (44.50mmol) were added to an eggplant-shaped bottle and dissolved in anhydrous chloroform. Activated for 2h in an ice bath, 1,3-dihydroxyacetone (11.70mmol) was added to the above reaction solution, and reacted at room temperature for 12h. The above reactions were all carried out under N2 protection. The reaction solution was spin-dried, and 200mL of anhydrous dichloromethane was added, and it was placed in a -20℃ refrigerator and frozen for 4h. After crystallization, a white solid product was obtained by suction filtration.

[0057] The above white solid product (9.65mmol) was dissolved in tetrahydrofuran: benzene (70mL: 14mL), and 7mL of distilled water was added under stirring. Sodium borohydride (36.21mmol) was added under ice bath, and 4.11mL of glacial acetic acid was added to terminate the reaction after stirring for 0.5h, and 70mL of anhydrous chloroform was added to the reaction solution. The above reaction solution was washed twice with 5% sodium bicarbonate solution and once with saturated sodium chloride solution, and the organic layer was separated by extraction. An appropriate amount of anhydrous sodium sulfate was added to the organic layer to remove moisture, and anhydrous sodium sulfate was removed by suction filtration. After removing the organic solvent with a rotary evaporator, a white solid product was obtained.

[0058] 4-Dimethylaminobutyric acid hydrochloride (17.95mmol), DMAP (10.14mmol), EDCI (22.43mmol) were added to an eggplant-shaped bottle and dissolved with anhydrous chloroform. Activated for 2h under ice bath, and the above white solid product (8.97mmol) was added to the above reaction solution and reacted at room temperature for 12h. The above reactions were all carried out under N2 protection. Compound 3-TG-18 was obtained by separation and purification by silica gel column chromatography.

[0059] Example 7

[0060] Oleic acid (35.40 mmol), DMAP (17.80 mmol), and EDCI (35.40 mmol) were added to an eggplant-shaped flask and dissolved with anhydrous dichloromethane. Activated for 2 h in an ice bath, 1,3-dihydroxyacetone (15.80 mmol) was added to the above reaction solution and reacted at room temperature for 12 h. The above reactions were all carried out under N2 protection. After separation and purification by silica gel column chromatography, a colorless oily product was obtained.

[0061] The colorless oily product (9.44 mmol) was dissolved in tetrahydrofuran: benzene (40 mL: 8 mL), and 7 mL of distilled water was added under stirring. Sodium borohydride (35.16 mmol) was added under ice bath, and 3.99 mL of glacial acetic acid was added to terminate the reaction after stirring for 0.5 h, and 40 mL of anhydrous chloroform was added to the reaction solution. The reaction solution was washed twice with 5% sodium bicarbonate solution and once with saturated sodium chloride solution, and the organic layer was separated by extraction. An appropriate amount of anhydrous sodium sulfate was added to the organic layer to remove moisture, and the anhydrous sodium sulfate was removed by suction filtration. After removing the organic solvent with a rotary evaporator, a colorless oily product was obtained.

[0062] 4-Dimethylaminobutyric acid hydrochloride (17.00 mmol), DMAP (9.61 mmol), EDCI (21.25 mmol) were added to an eggplant-shaped bottle and dissolved with anhydrous chloroform. Activated for 2 h under ice bath, and the above colorless oily product (8.50 mmol) was added to the above reaction solution and reacted at room temperature for 12 h. The compound 3-TG-OA was obtained by separation and purification by silica gel column chromatography.

[0063] Example 8: Release of lipid nanoparticles prepared from triglyceride-like ionizable lipids

[0064] The degradability of ionizable lipids is an important factor affecting the application of lipids. Therefore, examining the release of ionizable lipids is helpful to predict the degradability of lipids in vivo.

[0065] The lipid nanoparticles were dispersed in phosphate buffered saline (PBS, pH 7.4) with or without 1000 IU lipase, incubated at 37°C, 100 rpm in a shaker for 24 h, and centrifuged at 13000 rpm. The free siRNA concentration in the supernatant was detected and calculated using an ELISA reader to obtain the release rate of the lipid nanoparticles.

[0066] The results are as follows Figure 1 As shown. Under the condition of adding lipase, the lipid nanoparticles prepared by triglyceride-like ionizable lipids released more siRNA compared with MC3-LNPs, among which 2-TG-LA-LNPs released the largest amount, indicating that lipase promoted the cleavage of ester bonds in the triglyceride-like backbone, allowing siRNA to be released quickly. Therefore, triglyceride-like ionizable lipids have higher degradability.

[0067] Example 9: Lysosomal escape of lipid nanoparticles prepared from triglyceride-like ionizable lipids

[0068] The lysosomal escape efficiency of ionizable lipids is an important factor affecting the application of lipids.

[0069] 4×10 5RAW264.7 cells were seeded in a 15 mm confocal dish at a density of 100 cells / well and placed in a cell culture incubator for 12 hours to allow the cells to adhere to the wall. Then, the old culture medium was aspirated with a pipette, and lipid nanoparticles (siRNA, 200 nM) diluted with serum-free medium were added to each well, and then placed in a cell culture incubator for incubation for 6 hours. After the incubation, the drug-containing culture medium was discarded and washed three times with cold PBS at pH 7.4. Lysosomes were stained green with a lysosomal fluorescent probe (LysoTrackerGreen DND-26) and washed three times with PBS at pH 7.4. The cell nuclei were stained blue with Hoechst 33342 and washed three times with PBS at pH 7.4. After adding 1 mL of PBS (pH 7.4) to the confocal dish, the co-localization of lipid nanoparticles and lysosomes was observed using a laser confocal microscope.

[0070] The results are as follows Figure 2 The results showed that 2-TG-LA-LNPs had a lower Pearson colocalization coefficient than MC3-LNPs and thus had a higher lysosomal escape efficiency.

[0071] Example 10: Study on the gene silencing effect of lipid nanoparticles in vivo

[0072] Mice (C57BL / 6, purchased from the Animal Experiment Center of Shenyang Pharmaceutical University) were randomly divided into groups, with 4 mice in each group. LPS / D-GalN (model group) and Untreated (normal group) were used as control groups. The drug-treated groups were given magnesium isoglycyrrhizinate (MgIG) injection (4 mg / kg), free siRNA, 2-TG-LA-LNPs and MC3-LNPs (siRNA, 0.75 mg / kg) via the tail vein. 24 hours after administration, except for the normal group, 200 μL of a mixed solution of lipopolysaccharide (10 μg / kg) and D-GalN (300 mg / kg) was intraperitoneally injected to establish the mouse ALF model. After 5 hours of induction, the mice were killed. The level of serum IL-1β was detected using a mouse ELISA kit.

[0073] The results are as follows Figure 3 The results showed that both the 2-TG-LA-LNPs group and the MC3-LNPs group could effectively reduce the level of IL-1β in mouse serum, indicating that both lipid nanoparticles could effectively achieve gene silencing in vivo, among which 2-TG-LA-LNPs had a better gene silencing effect and was significantly different from MC3-LNPs.

[0074] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A triglyceride-like ionizable lipid, characterized in that: Its structural formula is as follows: wherein n is 2, 3 or 4, R1 and R2 are each independently selected from -(CH2)7CH=CHCH2CH=CH(CH2)4CH3, -(CH2)7CH=CH(CH2)7CH3, -(CH2) 12 CH3, -(CH2) 14 CH3 or -(CH2) 16 CH3.

2. The triglyceride-like ionizable lipid according to claim 1, characterized in that The tertiary amine head group is N,N-dimethyl tertiary amine, and is located at the β-, γ- or δ-position of the ester bond, respectively; the connecting region is triglyceride; and the fatty acid tail chain is myristic acid, palmitic acid, stearic acid, oleic acid or linoleic acid.

3. The triglyceride-like ionizable lipid according to claim 1 or claim 2, characterized in that Selected from the following compounds: 2-TG-LA 3-TG-LA 4-TG-LA 3-TG-14 3-TG-16 3-TG-18 3-TG-OA 4. The method for preparing the triglyceride-like ionizable lipid according to any one of claims 1 to 3, characterized in that: The preparation method comprises the following steps: Under the action of a catalyst, 1,3-dihydroxyacetone is connected to fatty acids through an esterification reaction and a triglyceride-like tail structure is obtained through reduction. The tail structure is connected to an acid containing a tertiary amino head through an esterification reaction to obtain the triglyceride-like tail structure. The catalysts are 4-dimethylaminopyridine (DMAP) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI).

5. Use of the triglyceride-like ionizable lipid according to any one of claims 1 to 3 in the preparation of a nucleic acid drug delivery vector.

6. The use according to claim 5, characterized in that: The delivery vector is used for the delivery of one or more of pDNA, siRNA, ASO or mRNA.

7. The use according to claim 5, characterized in that: The delivery vehicle is a lipid nanoparticle.

8. The use according to claim 5, characterized in that: The nucleic acid drug delivery vector is locally administered via microneedle, injection or perfusion in the muscle, subcutaneously, intradermally or intratumorally, or the nucleic acid drug delivery vector is administered via intravenous injection.

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