A PEG lipid compound and its application

Abstract

The present invention discloses a PEG lipid compound and its application. A PEG lipid compound, the structure of which is shown in formula (I), wherein x is selected from a positive integer of 11 to 17. The PEG lipid compound of the present invention has good biocompatibility and excellent in vivo mRNA delivery efficiency. The PEG lipid compound of the present invention has high synthesis efficiency and low cost, and is easy to scale up for commercial application. The delivery effect of the PEG lipid compound of the present invention corresponding to the LNP formula exceeds that of the commercial ionizable lipid SM-102 and the commercial PEG lipid DMG-PEG2000.

Description

Technical Field

[0001] The invention belongs to the field of new medicinal materials and relates to a PEG lipid compound and application thereof. Background Art

[0002] Compared with conventional vaccines, mRNA vaccines have the advantages of low cost, high production efficiency, high safety, and the potential to synthesize any protein. Therefore, they have great application potential for new infectious viruses that traditional vaccines are unable to deal with. However, due to the instability of mRNA molecules, easy degradation by RNases, and low in vivo delivery efficiency, the application of mRNA vaccines has been limited. To achieve the widespread application of mRNA vaccines, it is necessary to focus on solving the problem of delivery technology. mRNA vaccines need to have a suitable delivery carrier to deliver them into the body in order to have a better immune effect, so the development of an efficient and stable delivery system is the key to the success of mRNA vaccines.

[0003] The advanced delivery carrier of mRNA is lipid nanoparticle (LNP), which usually contains at least ionizable lipids, neutral auxiliary lipids and phospholipid polyethylene glycol derivatives. Among them, phospholipid polyethylene glycol derivatives play a key role in the particle size control, in vivo and in vitro stability and delivery effect of LNP. At the same time, the synthesis steps of phospholipid polyethylene glycol derivatives are many, the production cost is high, and it is the compound with the highest unit price among LNP components. Summary of the invention

[0004] The object of the present invention is to provide a PEG lipid compound and application thereof in view of the above-mentioned deficiencies in the prior art.

[0005] Another object of the present invention is to provide a lipid nanoparticle composition.

[0006] Another object of the present invention is to provide application of the lipid nanoparticle composition.

[0007] The purpose of the present invention can be achieved through the following technical solutions:

[0008] A PEG lipid compound, the structure of which is shown below:

[0009] ,

[0010] Wherein x is selected from a positive integer between 11 and 17.

[0011] As a preferred embodiment of the present invention, the x is selected from 11, 13, 15, and 17.

[0012] As further preferred embodiment of the present invention, x is selected from 11.

[0013] The preparation route of the compound of the present invention is as follows:

[0014]

[0015] The use of the PEG lipid compound in the preparation of nanolipid particles is preferably used in the preparation of nanolipid particles for delivering nucleic acids, and further preferably used in the preparation of nanolipid particles for delivering RNA or mRNA.

[0016] A lipid nanoparticle composition contains the PEG lipid compound of the present invention.

[0017] As a preferred embodiment of the present invention, the lipid nanoparticle composition further comprises one or more of ionizable lipids and neutral lipids.

[0018] As a preferred embodiment of the present invention, the neutral lipid is selected from any one or two of DOPE and cholesterol.

[0019] As a preferred embodiment of the present invention, the ionizable lipid is ionizable lipid H-1:

[0020] .

[0021] As a preferred embodiment of the present invention, the lipid nanoparticle composition consists of H-1 ionizable lipid compound H-1, DOPE, cholesterol and the PEG lipid compound of the present invention.

[0022] As a further preferred embodiment of the present invention, the molar ratio of the ionizable lipid compound H-1:DOPE:cholesterol:the PEG lipid compound of the present invention is 45~55:10~15:35~40:1~3, preferably 50:10:38.5:1.5.

[0023] The invention relates to the use of the lipid nanoparticle composition in the preparation of mRNA drugs or vaccines.

[0024] Beneficial effects:

[0025] The PEG lipid compound of the present invention has good biocompatibility and excellent in vivo mRNA delivery efficiency.

[0026] The PEG lipid of the present invention is synthesized by a one-step method, and has a simple synthesis process, high synthesis efficiency, low production cost, and is convenient for commercial production.

[0027] Its unique secondary amine structure and single lipid bond alkyl chain can enhance the binding of LNP to mRNA, and have better encapsulation rate and delivery effect; at the same time, the PEG lipid of the present invention forms a proton sponge effect in the animal cell endosomes, enhances the release of mRNA-LNP from the endosomes, and improves the in vivo delivery ability of mRNA. The delivery effect of the ionizable lipid compound of the present invention corresponding to the LNP formula exceeds that of the commercial ionizable lipid SM-102 and the commercial PEG lipid DMG-PEG2000. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 HNMR spectrum of PEG lipid compound P-1 (CDCl 3 solution).

[0029] Figure 2 HNMR spectrum of PEG lipid compound P-2 (CDCl 3 solution).

[0030] Figure 3 HNMR spectrum of PEG lipid compound P-3 (CDCl 3 solution).

[0031] Figure 4 HNMR spectrum of PEG lipid compound P-4 (CDCl 3 solution).

[0032] Figure 5 The mRNA lipid nanoparticles No. 1-7 prepared in Example 5 were injected into the mouse muscle and photographed by animal imaging equipment. DETAILED DESCRIPTION

[0033] Example 1 Synthesis of PEG lipid compound P-1:

[0034] 10mmol methoxypolyethylene glycol amine (MW2000), 30mmol dodecyl acrylate and 30mL dimethyl sulfoxide (DMSO) were added to a 50mL reaction bottle equipped with a magnetic rod. The reaction tube was heated and stirred at 90°C for 72 hours. After the reaction was complete under TLC monitoring, the reaction was cooled to room temperature and the solvent was removed by a rotary evaporator. The product was separated by a thin layer chromatography column (silica gel column, the eluent was dichloromethane: methanol: triethylamine volume ratio = 90:9:1), and a white solid P-1 was obtained with a yield of 89%. The specific reaction formula is as follows:

[0035]

[0036] The H NMR spectrum of ionizable lipid compound P-1 is shown in Figure 1 .

[0037] Example 2 Synthesis of PEG lipid compound P-2:

[0038] 10mmol methoxypolyethylene glycol amine (MW2000), 30mmol tetradecyl acrylate and 30mL dichloromethane were added to a 50mL reaction bottle equipped with a magnetic rod. The reaction tube was heated and stirred at 85°C for 72 hours. After the reaction was complete under TLC monitoring, the reaction was cooled to room temperature and the solvent was removed by a rotary evaporator. The product was separated by a thin layer chromatography column (silica gel column, eluent was dichloromethane: methanol: triethylamine volume ratio = 90:9:1), and a reddish brown solid P-2 was obtained with a yield of 87%. The specific reaction formula is as follows:

[0039]

[0040] The H NMR spectrum of ionizable lipid compound P-2 is shown in Figure 2 .

[0041] Example 3 Synthesis of PEG lipid compound P-3:

[0042] 10mmol methoxypolyethylene glycol amine (MW2000), 30mmol hexadecyl acrylate and 30mL dichloromethane were added to a 50mL reaction bottle equipped with a magnetic rod. The reaction tube was heated and stirred at 90°C for 72 hours. After the reaction was complete under TLC monitoring, the reaction was cooled to room temperature and the solvent was removed by a rotary evaporator. The product was separated by a thin layer chromatography column (silica gel column, eluent was dichloromethane: methanol: triethylamine volume ratio = 90:9:1), and a reddish brown solid P-3 was obtained with a yield of 86%. The specific reaction formula is as follows:

[0043]

[0044] The H NMR spectrum of PEG lipid compound P-3 is shown in Figure 3 .

[0045] Example 4 Synthesis of PEG lipid compound P-4:

[0046] 10mmol methoxypolyethylene glycol amine (MW2000), 30mmol octadecyl acrylate and 30mL dichloromethane were added to a 50mL reaction bottle equipped with a magnetic rod. The reaction tube was heated and stirred at 95°C for 84 hours. After the reaction was complete under TLC monitoring, the reaction was cooled to room temperature and the solvent was removed by a rotary evaporator. The product was separated by a thin layer chromatography column (silica gel column, eluent was dichloromethane: methanol: triethylamine volume ratio = 90:9:1), and a dark brown solid P-4 was obtained with a yield of 84%. The specific reaction formula is as follows:

[0047]

[0048] The H NMR spectrum of ionizable lipid compound P-4 is shown in Figure 4 .

[0049] Example 5: Preparation and detection of lipid nanoparticles (LNP preparations)

[0050] S1. Prepare a 5 mM pH 3.0 citrate buffer solution containing firefly luciferase (fluc) mRNA as the aqueous phase.

[0051] S2, prepare anhydrous ethanol solution of SM-102 ionizable lipid and H1 ionizable lipid, DOPE, cholesterol, DMG-PEG 2000 and PEG lipid compounds P1-P4 of the present invention. Prepare a lipid mixed solution as an organic phase according to the molar ratio of ionizable lipid compound lipid: DOPE: cholesterol: PEG lipid of 50:10:38.5:1.5.

[0052] S3, through the microfluidic device, the above two solutions are mixed in the microfluidic chip, the aqueous and organic phase solutions enter from the two sides and the middle flow path of the microfluidic chip respectively, the total flow rate of the aqueous phase is controlled at 3ml / min, the organic phase flow rate is controlled at 1ml / min, the aqueous phase: organic phase flow rate ratio is 3:1, the two phases flow path converge in the chip and then mix, the weight ratio of total lipids to mRNA is about 15~30:1, and mRNA lipid nanoparticles (mRNA-LNP) are prepared by combining positively charged lipids with negatively charged mRNA.

[0053] S4, the obtained mRNA-LNP needs to be diluted 10 times with 10mM pH7.0 PBS buffer and purified by ultrafiltration concentration through an ultrafiltration tube with a pore size of 100KD filter membrane. The ultrafiltration method is a 30-degree fixed angle rotor, a centrifugal force of 2000g, and centrifugation at room temperature of 25°C to concentrate to 1 / 10 volume.

[0054] S5, mRNA-LNP was filtered through a 0.22 μm filter membrane and stored at 2-8°C.

[0055] S6. The average particle size and polydispersity index (PDI) of the mRNA-LNP samples were determined using a dynamic light scattering nanoparticle size analyzer. The test results are shown in Table 1.

[0056] S7, Quant-it Ribogreen RNA quantification kit (Thermo

[0057] Fisher Scientific, UK) to determine the encapsulation efficiency of lipid nanoparticles. The specific operation is:

[0058] (1) Dilute the prepared mRNA-LNP suspension and PBS (negative control, equal volume of TE buffer) to 4 ng / μl using the TE buffer in the kit to obtain the mRNA-LNP working solution.

[0059] (2) Continue to use TE buffer (or TE buffer containing 2% Triton-X100) to further dilute the mRNA-LNP working solution with equal volume, mix well and let it stand at 37°C for 10 min (TE buffer without Triton-X100 is used to measure the unencapsulated free mRNA, while TE buffer containing 2% Triton-X100 is used to measure the total mRNA in the mRNA-LNP working solution, which includes free mRNA and mRNA encapsulated in lipid nanoparticles).

[0060] (3) After calibrating the standard curve of fluorescence intensity: concentration with the standard substance, pipette an appropriate amount of Quant-it™ RiboGreen RNA reagent nucleic acid dye into each group of samples according to the kit instructions, stain for 5 minutes, transfer each group of stained samples to the microplate reader for detection, and use the software to accurately quantify the mRNA in the sample.

[0061] (4) The mRNA encapsulation efficiency in lipid nanoparticles was calculated using the following formula:

[0062] Encapsulation efficiency = [1-m (free mRNA): m (total mRNA)] × 100%, test results are shown in Table 1.

[0063] Table 1. Test results of physical parameters of lipid nanoparticles

[0064] .

[0065] Example 6 Verification of the delivery system effect through in vivo animal experiments

[0066] The LNP-1-7 mRNA lipid nanoparticles prepared in Example 5 and the control sample were injected into the leg muscles of mice at the same time, with an injection dose of 5 μg mRNA / mouse. After 6 hours, the delivery effect was detected by taking pictures with an animal imaging device and calculating the fluorescence intensity of the injection site. The test results are shown in Table 2 and Figure 5 .

[0067] Table 2. In vivo experimental delivery effect

[0068] .

[0069] According to the data in Table 1, the particle size and encapsulation efficiency of mRAN-LNP prepared by combining the ionizable lipid compound H1 with PEG lipids P-1, P-2, P-3, and P-4 reached the level of commercial PEG lipid DMG-PEG 2000, and were partially superior to DMG-PEG 2000. The particle size and encapsulation efficiency of mRAN-LNP prepared by combining the commercial ionizable lipid compound SM-102 with PEG lipid P-1 were superior to those of commercial PEG lipid DMG-PEG 2000. Due to the secondary amine structure and lipid bond in PEG lipid P-1, PEG lipid P-1 has more positive charge than DMG-PEG 2000, which can significantly improve the encapsulation efficiency.

[0070] According to the data in Table 2, the in vivo delivery effect of mRAN-LNP prepared by combining ionizable lipid compound H1 with PEG lipids P-1, P-2, P-3, and P-4 exceeds the level of commercial PEG lipid DMG-PEG 2000, among which the effect of ionizable lipid compound H1 combined with PEG lipid P-1 is significantly better than that of ionizable lipid compound H1 combined with DMG-PEG 2000. The in vivo delivery effect of mRAN-LNP prepared by combining commercial ionizable lipid compound SM-102 with PEG lipid P-1 is significantly better than that of commercial PEG lipid DMG-PEG 2000. The single-lipid alkyl chain of PEG lipid P-1 can make the PEG lipid fall off the LNP faster in the body circulation, improve the endocytosis of LNP, and thus have a better delivery effect.

Claims

1. A PEG lipid compound, characterized in that The structure is as follows: ， Wherein x is selected from a positive integer between 11 and 17.

2. The PEG lipid compound according to claim 1, characterized in that x is selected from 11, 13, 15, and 17.

3. The PEG lipid compound according to claim 1, characterized in that x is selected from 11.

4. The use of the PEG lipid compound described in any one of claims 1 to 3 in the preparation of nano lipid particles.

5. The use according to claim 4, characterized in that: Use of the PEG lipid compound according to any one of claims 1 to 3 in the preparation of nanolipid particles for delivering nucleic acids.

6. A lipid nanoparticle composition, characterized in that: Contains the PEG lipid compound according to any one of claims 1 to 3.

7. The lipid nanoparticle composition according to claim 6, characterized in that The lipid nanoparticle composition further comprises one or more of ionizable lipids and neutral lipids.

8. The lipid nanoparticle composition according to claim 7, characterized in that The neutral lipid is selected from any one or two of DOPE and cholesterol.

9. The lipid nanoparticle composition according to claim 7, characterized in that The ionizable lipid is ionizable lipid H-1: 。 10. The lipid nanoparticle composition according to claim 9, characterized in that The lipid nanoparticle composition consists of ionizable lipid H-1, DOPE, cholesterol and the PEG lipid compound described in any one of claims 1 to 3.

11. The lipid nanoparticle composition according to claim 10, characterized in that The molar ratio of ionizable lipid H-1:DOPE:cholesterol:PEG lipid compound according to any one of claims 1 to 3 is 45-55:10-15:35-40:1-3.

12. The lipid nanoparticle composition according to claim 11, characterized in that The molar ratio of ionizable lipid H-1:DOPE:cholesterol:PEG lipid compound according to any one of claims 1 to 3 is 50:10:38.5:1.

5.

13. Use of the lipid nanoparticle composition according to any one of claims 6 to 12 in the preparation of mRNA drugs or vaccines.

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