Neutral lipid for mRNA delivery, two-component lipid nanoparticle, preparation method and application

Abstract

The invention relates to the technical field of biological medicine, and discloses a neutral lipid for mRNA delivery, a two-component lipid nanoparticle, a preparation method and application, and the preparation method comprises the following steps: the neutral lipid is obtained through a Ugi reaction of amine, carboxylic acid, aldehyde and isocyanide compounds; wherein the amine is polyethylene glycol-terminated amine, the carboxylic acid is a uracil carboxylic acid derivative, the aldehyde is one of C12, C18 and oleic acid chain aldehydes, and the isocyanide compound is one of C12, C18 and oleic acid chain isocyanide compounds; according to the neutral lipid structure prepared by the invention, a basic group complementary pairing delivery mechanism of uracil and polyA tail of mRNA is used for replacing a traditional model of combining electrostatic interaction of cationic lipid with mRNA, so that the problem of related inflammatory toxicity caused by cationic charges existing in ionized lipid is solved. Neutral lipid and cholesterol form novel two-component LNP, and efficient delivery of mRNA can be achieved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a neutral lipid for mRNA delivery, a two-component lipid nanoparticle, a preparation method and application. BACKGROUND

[0002] Lipid nanoparticles (LNP) as the most mature non-viral gene delivery system is an important material in the process of vaccine preparation. A typical LNP is usually composed of four key components: ionizable lipid, neutral phospholipid, cholesterol and polyethylene glycol (PEG) lipid to achieve efficient encapsulation of mRNA, stable transportation in vivo and cell delivery. However, there is a delicate dynamic balance and mutual restraint among the four components. Adjusting the proportion of any single component may trigger a chain reaction, affecting particle formation, stability, delivery efficiency and even in vivo distribution, making the systematic optimization of the formulation a highly complex engineering challenge. Among them, ionizable lipid efficiently binds and compresses negatively charged mRNA molecules through electrostatic interaction, which is the core component of LNP. Although ionizable lipid can effectively deliver mRNA in vivo, the LNP containing ionizable lipid has problems of insufficient systemic safety and extrahepatic tissue targeting ability. On the one hand, ionizable lipid and exogenous mRNA can cause the release of pro-inflammatory cytokines (such as TNF-α, IFN-γ, IL-6, IL-1) by activating pattern recognition receptors (such as Toll-like receptors TLR) of innate immunity. Although this inflammatory response may be beneficial to therapeutic tumor vaccines, the high immune stimulation limits its application in preventive vaccines and protein replacement therapy. Especially in applications requiring multiple doses, highly pro-inflammatory LNP not only may interfere with mRNA expression efficiency, but also may cause cumulative tissue damage. On the other hand, LNP based on ionizable lipid is easily adsorbed and interacted with various serum proteins in the blood due to its surface properties (such as charge, PEG density) when administered systemically. The formation of this "protein crown" makes LNP mainly taken up by hepatocytes and expressed by the low-density lipoprotein receptor pathway in the liver, resulting in high accumulation of nucleic acid drugs in the liver. This strong liver tropism is beneficial for the treatment of liver-related diseases, but greatly limits the effective delivery of LNP to extrahepatic tissues, hindering the clinical application potential of mRNA in a wider range of diseases. SUMMARY

[0003] The present application provides a neutral lipid for mRNA delivery, a two-component lipid nanoparticle, a preparation method and application to solve the problems in the prior art.

[0004] The technical scheme adopted by the present application is: a preparation method of a neutral lipid for mRNA delivery, comprising the following steps: the neutral lipid is obtained by Ugi reaction of amine, carboxylic acid, aldehyde and isonitrile compound;

[0005] Wherein the amine is a polyethylene glycol end amine, the carboxylic acid is a uracil carboxylic acid derivative, the aldehyde is one of C12, C18 and oleic acid chain aldehyde, and the isonitrile compound is one of C12, C18 and oleic acid chain isonitrile compound.

[0006] Further, the preparation process of the isonitrile compound is as follows:

[0007] The fatty amine is refluxed and stirred with methyl formate at 60 DEG C.

[0008] Triethylamine is added to the reaction solution, and phosphorus oxychloride is added dropwise, and the reaction is carried out at-20 DEG C to obtain the product.

[0009] Further, the preparation process of the uracil carboxylic acid derivative is as follows:

[0010] 5-Hydroxymethyluracil and mercaptoacetic acid are fully reacted in an acid solution to obtain the desired uracil carboxylic acid derivative.

[0011] Further, the preparation process of the aldehyde is as follows:

[0012] Anhydrous dichloromethane, primary alcohol and pyridinium chlorochromate are fully reacted to obtain the desired aldehyde.

[0013] A neutral lipid for mRNA delivery, the neutral lipid is electrically neutral.

[0014] A two-component lipid nanoparticle obtained by a neutral lipid for mRNA delivery, the lipid nanoparticle is prepared by a microfluidic method; wherein the molar ratio of the neutral lipid and cholesterol is 5-6:4-5.

[0015] A preparation method of a two-component lipid nanoparticle for mRNA delivery, the preparation process is as follows:

[0016] The neutral lipid and cholesterol are dissolved in a solvent as an organic phase; the mRNA is dissolved in water as an aqueous phase; and the lipid nanoparticle is obtained by a microfluidic method.

[0017] The application of a two-component lipid nanoparticle for mRNA delivery, the lipid nanoparticle is applied in vaccine preparation.

[0018] The beneficial effects of the present application are:

[0019] The neutral lipid structure prepared by the application replaces the traditional cationic lipid electrostatic interaction binding mRNA model with the base complementary pairing delivery mechanism of uracil and the polyA tail of mRNA, solves the related inflammatory toxicity caused by the cationic charge in the ionized lipid. And the neutral lipid and cholesterol form a new two-component LNP, which blocks the ApoE-mediated liver cell uptake path, and realizes efficient delivery of mRNA. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 The H NMR spectrum of the neutral lipid obtained in embodiment 1 of the application. 1 H NMR spectrum.

[0021] Figure 2 The HRMS diagram of the neutral lipid obtained in embodiment 1 of the application.

[0022] Figure 3 The performance test results of the LNP obtained in embodiments 1, 2 and 3 of the application, A is a particle size schematic diagram, B is a PDI diagram, C is a Zeta potential, and D is the Zeta potential of the LNP obtained in embodiment 1 and the LNP obtained from the commercially available SM-102 lipid.

[0023] Figure 4 The in vivo expression of the LNP obtained in embodiment 1 of the application and the LNP obtained from the commercially available SM-102 lipid in delivering 5ug luc mRNA in Balb / c mice.

[0024] Figure 5 The cell survival rate results obtained by the CCK-8 kit after adding the LNP obtained in embodiment 1 of the application and the LNP obtained from the commercially available SM-102 lipid to DC cells for 24 hours.

[0025] Figure 6 The Elisa experiment results of the LNP obtained in embodiment 1 of the application and the LNP obtained from the commercially available SM-102 lipid, A is the level of key pro-inflammatory factors in serum, and B is the H&E staining result of the main organs. DETAILED DESCRIPTION

[0026] The application will be further described below in combination with the drawings and specific embodiments.

[0027] A preparation method of a neutral lipid for mRNA delivery, comprising the following steps: the neutral lipid is an amine, carboxylic acid, aldehyde, isonitrile compound obtained by Ugi reaction; the reaction process is as follows: the above raw materials are dissolved in dimethylformamide in a molar ratio of 1:1:1:1, and reacted at 60 DEG C for 24 hours, and then subjected to silica gel column chromatography (dichloromethane / methanol gradient elution) to obtain the neutral lipid compound.

[0028]

[0029] The amine is a polyethylene glycol-terminated amine;

[0030]

[0031] The carboxylic acid is a uracil carboxylic acid derivative;

[0032]

[0033] Aldehydes are one of the C12, C18, and oleic acid chain aldehydes;

[0034]

[0035] The isonitrile compound is one of the C12, C18 and oleic acid chain isonitrile compounds;

[0036]

[0037] The preparation process of isonitrile compounds is as follows:

[0038]

[0039] 54 mmol of aliphatic amine was dissolved in 100 mL of ethyl formate and refluxed at 60 °C with stirring for 20 h. After the reaction was complete, volatile components were removed by vacuum distillation, and a white solid residue was dissolved in dichloromethane. 20 mL of triethylenediamine was slowly added to the mixture at -20 °C, followed by dropwise addition of 54 mmol of phosphorus oxychloride POCl3. The reaction was maintained at low temperature for 2 h, then moved to room temperature and stirred for another 24 h. After terminating the reaction, the mixture was poured into 20 mL of deionized water cooled in an ice-water bath and extracted three times with dichloromethane. The combined organic phases were washed three times with saturated sodium chloride solution and dried over anhydrous ammonium sulfate. After removing the solvent by rotary evaporation, the product was purified by silica gel column chromatography to obtain the target product.

[0040] The preparation process of uracil carboxylic acid derivatives is as follows:

[0041]

[0042] 21.7 mmol of 5-hydroxymethyluracil and 21.7 mmol of mercaptoacetic acid were dissolved in 2N hydrochloric acid aqueous solution and reacted with stirring at 50 °C for 48 h. After the reaction was completed, the precipitate was collected by vacuum filtration through a Buchner funnel, and the white crystalline product was dried in a vacuum drying oven for 12 h to obtain the target compound.

[0043] The preparation process of aldehydes is as follows:

[0044]

[0045] Wastewater, dichloromethane, and primary alcohol were sequentially added to a drying reactor, followed by PCC powder (1.5 equivalents). The mixture was stirred at room temperature for 12 hours. After the reaction was complete, the solution was diluted with diethyl ether, filtered to remove the chromium salt precipitate, and the filtrate was washed sequentially with saturated sodium carbonate aqueous solution and saturated brine. The solution was dried over anhydrous magnesium sulfate, concentrated under reduced pressure, and the product was obtained by column chromatography.

[0046] Example 1

[0047] Neutral lipids are prepared according to the following procedure:

[0048] Uracil carboxylic acid, aminoPEG (P7) with a degree of polymerization of 7, octadecaldehyde, and dodecanoic acid (I12) were mixed in dimethylformamide in a molar ratio of 1:1:1:1 and reacted at 60°C for 24 hours. The resulting lipid molecule was denoted as U-P7-A. 18 -I 12 The structure is as follows:

[0049]

[0050] The prepared neutral lipids and cholesterol were dissolved in anhydrous ethanol at a molar ratio of 50:50, and mRNA was dissolved in pure water. Using microfluidic technology, the organic phase and aqueous phase were mixed at a mass ratio of 40:1 (lipids:mRNA) and a flow rate ratio of 3:1 (total flow rate of 12 mL / min) to prepare LNP.

[0051] The NMR results of the neutral lipids obtained in this embodiment are as follows: Figure 1 As shown, the high-resolution mass spectrometry (HRMS) results are as follows: Figure 2 As shown.

[0052] 1H-NMR:(DMSO-d6,400MHz,):δ(ppm)0.88(t,6H,-CH2CH3),1.22(s,50

[0053] H,-(CH2)14CH3,-NH-CH2-(CH2)10-CH3),3.20(t,2H,-NH-CH2-),3.23(m,7H,-CH2-S-CH2 -,-N-CH2-CH2-O,-O-CH2-CH3),3.42(m,4H,-CH2-S-CH2-,-N-CH2-CH2-O-),3.50(t,28H,

[0054] -O-(CH2-CH2)7-O),4.11(s,1H,-N-CH-CO-),7.95(s,1H,uracil,NH-CH-C),8.00(s,1H,

[0055] -CO-N-CH2-), 10.70(s,1H,-NH-CO-NH), 11.15(s,1H,-NH-CO-NH).

[0056] HRMS: [M+Na]+: 1067.7269, found: 1067.7262.

[0057] Example 2

[0058] The preparation process for neutral lipids is as described in Example 1. The difference lies in the fact that, in the preparation of LNP, the molar ratio of neutral lipids to cholesterol is 60:40.

[0059] Example 3

[0060] The preparation process for neutral lipids is as described in Example 1. The difference lies in the fact that, in the preparation of LNP, the molar ratio of neutral lipids to cholesterol is 40:60.

[0061] Figure 3 The figures show the test results of LNPs obtained in Examples 1, 2, and 3. As can be seen from the figures, the LNP obtained in Example 3 has a slightly larger particle size and PDI than those in Examples 1 and 2. The LNP obtained in Example 1 has a Zeta potential infinitely close to 0, and its surface is composed mainly of neutral lipids. The LNP obtained in Example 1 can form uniform and stable LNPs. The LNPs obtained in Examples 2 and 3, however, have slightly inferior performance.

[0062] from Figure 3 As can be seen from D, compared with the four-component LNP obtained by commercially available lipid SM-102, the two-component LNP obtained by this invention has a net charge of almost 0, while the LNP obtained by commercially available SM-102 has a significant positive charge.

[0063] The transfection efficiency experiment was conducted using the LNP obtained in Example 1, and the results are as follows: Figure 4 As shown in Table 1, the statistical results are as follows.

[0064] Table 1. In vivo expression of 5 μg luc mRNA delivered by different LNP systems in Balb / c mice

[0065]

[0066] from Figure 4 As shown in Table 1, in terms of in vivo targeted transfection, the LNPs obtained from the four-component SM-102 showed high aggregation in the liver, while the two-component LNPs showed higher targeting and transfection performance in the spleen.

[0067] The LNP obtained in Example 1 and the LNP obtained from commercially available SM-102 were subjected to in vivo safety testing. The test results are as follows: Figure 5As shown in the figure. The detection method involved adding different concentrations of LNP to DC cells, and then detecting cell viability using a CCK-8 assay kit after 24 hours. As can be seen from the figure, compared to the PBS control group and LNP obtained from commercially available SM-102, the LNP obtained in Example 1 maintained a cell viability of over 80% even at a high concentration of 7.5 μg / mL, indicating that it has lower cytotoxicity.

[0068] ELISA experiments were performed on the LNP obtained in Example 1 and the LNP obtained from commercially available SM-102. The results are as follows: Figure 6 As shown, the testing method was as follows: female Balb / c mice were injected with LNP containing 10 μg mRNA via the tail vein, and blood was collected from the orbital cavity 6 hours later. After centrifugation, the serum was collected for ELISA experiments.

[0069] As can be seen from the figure, mice injected with the LNP obtained in Example 1 had lower levels of typical pro-inflammatory cytokines in their serum, indicating a weaker acute inflammatory response induced by the LNP obtained in Example 1. Furthermore, histopathological analysis of major organs also showed that the LNP obtained in Example 1 did not cause significant tissue damage, demonstrating its good biocompatibility.

[0070] The neutral lipids obtained in this invention have hydrophilic air groups rich in hydroxyl groups, giving them excellent biocompatibility and the ability to mimic the long-cycle effect of PEG lipids. Furthermore, they simultaneously possess the structural stabilizing effect of neutral phospholipids and the long-cycle effect of PEG lipids. Therefore, by using neutral lipids to reduce the addition of PEG lipids and neutral phospholipids, LNP lipid nanoparticles are obtained.

[0071] The prepared LNP lipid nanoparticles utilize a base-complementary delivery mechanism between uracil and the polyA tail of mRNA to replace the traditional cationic lipid electrostatic interaction-based mRNA binding mode, thus avoiding the inflammatory toxicity caused by cationic charge. Neutral lipids block the ApoE-mediated hepatocyte uptake pathway, achieving highly efficient mRNA delivery in the spleen.

Claims

1. A method for preparing neutral lipids for mRNA delivery, characterized in that, Includes the following steps: Neutral lipids are obtained from amines, carboxylic acids, aldehydes, and isonitrile compounds via the Ugi reaction; The amine is a polyethylene glycol-terminated amine, the carboxylic acid is a uracil carboxylic acid derivative, the aldehyde is one of C12, C18 and oleic acid chain aldehydes, and the isonitrile is one of C12, C18 and oleic acid chain isonitrile compounds.

2. The method for preparing neutral lipids for mRNA delivery according to claim 1, characterized in that, The preparation process of the isonitrile compound is as follows: Fatty amines and methyl formate were refluxed and stirred at 60°C. The solution is prepared by adding triethylamine to the reaction solution, followed by the dropwise addition of phosphorus oxychloride, and allowing the reaction to proceed at -20°C until complete.

3. The method for preparing neutral lipids for mRNA delivery according to claim 1, characterized in that, The preparation process of the uracil carboxylic acid derivative is as follows: The desired uracil carboxylic acid derivative can be obtained by fully reacting 5-hydroxymethyluracil and mercaptoacetic acid in an acidic solution.

4. The method for preparing neutral lipids for mRNA delivery according to claim 1, characterized in that, The preparation process of the aldehydes is as follows: The desired aldehyde can be obtained by fully reacting anhydrous dichloromethane, a primary alcohol, and pyridinium chlorochromate.

5. The neutral lipid for mRNA delivery obtained by any of the preparation methods described in claims 1 to 4, characterized in that, The neutral lipid is electrically neutral.

6. A two-component lipid nanoparticle obtained using a neutral lipid for mRNA delivery as described in claim 5, characterized in that, Lipid nanoparticles were prepared using a microfluidic method, wherein the molar ratio of neutral lipids to cholesterol was 5–6:4–5.

7. The method for preparing two-component lipid nanoparticles for mRNA delivery as described in claim 6, characterized in that, The preparation process is as follows: Neutral lipids and cholesterol were dissolved in a solvent to form the organic phase; mRNA was dissolved in water to form the aqueous phase; and lipid nanoparticles were obtained using a microfluidic method.

8. The application of the two-component lipid nanoparticles for mRNA delivery as described in claim 6, characterized in that, Application of the lipid nanoparticles in vaccine preparation.

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