Asymmetric branched peg-modified lipid compound, preparation method therefor and use thereof

Abstract

Provided in the present invention are an asymmetric branched PEG-modified lipid compound, a preparation method therefor, the use thereof, and a nucleic acid delivery system containing same.

Description

Asymmetric branched PEG-modified lipid compounds, their preparation methods and applications Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to asymmetric branched PEG-modified lipid compounds, their preparation methods, their uses, and nucleic acid delivery systems containing them. Background Technology

[0002] Nucleic acid drugs include DNA, messenger RNA (mRNA), small interfering RNA (siRNA), microRNA (miRNA), aptamers, plasmids, etc. The application of nucleic acid drugs is limited by their chemical instability, such as degradation by nucleases, hydrolysis of phosphodiester bonds in the molecule, or the influence of temperature and pH, ultimately degrading them into individual nucleotides and losing their efficacy.

[0003] The application of nucleic acid drugs often requires special delivery carriers or systems for protection, such as polymeric complexes, small nucleic acid-modified N-acetylgalactosamine (GalNAc), and liposome nanoparticles (LNPs). Among these, LNPs are the most widely used carriers for nucleic acid delivery. The successful approval of mRNA-LNP COVID-19 vaccines has also made LNPs one of the most promising nucleic acid delivery systems, especially for large-molecule nucleic acid drugs such as mRNA or DNA. However, reports of allergic reactions during COVID-19 vaccination have focused public attention on the safety of LNPs as a delivery system. The US CDC has reported and warned of potential allergic or acute inflammatory adverse reactions after mRNA-LNP vaccination, and the US Food and Drug Administration (FDA) has added four black-box warnings to the mRNA-LNP vaccine package insert. Numerous studies have shown a positive correlation between adverse reactions after vaccination and the level of anti-PEG antibodies (anti-PEG IgG and anti-PEG IgM) produced by the body. Repeated exposure to LNP vaccines and other polyethylene glycol-containing preparations can enhance anti-PEG antibody levels. [1] Furthermore, with the increase in PEG-containing daily necessities, the level of pre-existing anti-PEG antibodies in the human body has also increased. Pre-existing anti-PEG antibodies preferentially bind to PEG-modified drugs, thereby accelerating drug clearance and reducing efficacy. Simultaneously, numerous cases have linked high expression of anti-PEG IgG and IgM to clinical trial failures. Given that billions of people worldwide have received multiple doses of mRNA-LNP vaccines over the past three years, the level of anti-PEG antibodies in the population may be significantly elevated, posing potential risks to the development and clinical research of PEG-modified drugs.

[0004] The mRNA-LNP system comprises four commonly used lipids (cationic / ionizable lipids, accessory lipids, cholesterol, and PEG-lipids) and RNA drugs. Ionizable lipids interact with negatively charged RNA to encapsulate nucleic acids. Accessory lipids and cholesterol both increase the biocompatibility of LNPs with cell membranes. Accessory lipids stabilize the structure of LNPs and improve drug loading efficiency. Cholesterol increases the rigidity and stability of LNPs, preventing them from disintegrating in the body and reducing the probability of recognition and clearance by the immune system, thus prolonging their half-life in vivo. As a lipid, cholesterol molecules themselves have good biocompatibility and play a crucial role in life processes. PEG-lipids can reduce particle aggregation, control particle size, and prolong half-life.

[0005] Despite the numerous benefits of PEG-lipids, their potential immunogenicity and the generation of anti-PEG antibodies after repeated injections have become pressing concerns. A growing body of research and clinical evidence suggests that PEG-lipids are associated with allergic reactions and a rapid decline in efficacy after repeated administration. [1-3] The immunogenicity of PEG-lipids in LNP nucleic acid delivery systems has once again attracted increasing attention from the industry. Solving the immunogenicity problem of PEG-lipids in LNP-based nucleic acid delivery systems is of great significance for improving the delivery efficiency, clinical safety, and efficacy of nucleic acid drugs based on LNP systems.

[0006] Currently, numerous studies focus on developing compounds that can replace PEG-lipids to achieve low immunogenicity. These alternatives include polymer molecules derived from PEG-based modifications and other non-PEG compounds. Common methods for modifying PEG-based molecules include altering the PEG chain length, changing the hydrophobic end bonds, copolymerizing with polypropylene glycol, or modifying with fatty acids. These synthetic routes are relatively complex, increasing production costs and time. Non-PEG compounds can be categorized into synthetic and natural molecules based on their preparation methods. Among these, polyamino esters... [4] Polyamino acids [5] and polyglycerol [6,7] While these technologies have shown some potential in nucleic acid delivery, most related studies focus on nucleic acid-polymer complexes, and their application in RNA-LNP requires further investigation. In addition, some natural polymers such as chitosan, hyaluronic acid, and sodium alginate... [8,9]Its application in nucleic acid delivery is also under development, but delivery efficiency and other parameters require further in vivo data for validation. Therefore, research on compounds that can replace PEG-lipids is currently at the forefront, but there is still a long way to go before practical clinical use. Since the immunogenicity mechanism of PEG is still under investigation, the experimental results of related alternatives require further theoretical support, as well as more in vivo experimental data to support their biocompatibility and safety. Therefore, it is still necessary to develop suitable alternatives to PEG-lipids to improve the safety and / or effectiveness of RNA-LNP delivery systems. Summary of the Invention

[0007] The present invention provides a lipid compound modified with asymmetric branching PEG to satisfy the aforementioned needs.

[0008] Specifically, the asymmetric branched PEG-modified lipid compound provided by this invention exhibits low immunogenicity and, upon repeated injection, induces significantly lower levels of anti-PEG antibodies compared to the market-controlled PEG lipid (DMG-PEG2k (dimyristicoyl-sn-glycerol-polyethylene glycol 2000), the PEG lipid used in Moderna's COVID-19 vaccine). LNPs prepared using this asymmetric branched PEG-modified lipid compound demonstrate significantly higher in vivo delivery / transfection efficiency compared to the market-controlled compound. Without being theoretically limited, it is believed that this higher in vivo delivery / transfection efficiency is precisely due to the induction of lower levels of anti-PEG antibodies in vivo.

[0009] In the asymmetric branched PEG-modified lipid compound provided by this invention, cholesterol molecules, acting as hydrophobic ends, are linked to asymmetric branched PEG molecules via polyamino organic acid linkers (cleavable linkers). As shown in Figure 1, asymmetric branched PEG refers to the presence of multiple PEG branches, each with a different length, thus exhibiting asymmetry.

[0010] Therefore, a first aspect of the present invention provides an asymmetric branched PEG-modified lipid compound having the following structural formula:

[0011] Asymmetric branched PEG-polyamino organic acid linker-cholesterol.

[0012] According to certain implementations, the asymmetric branched PEG can be an asymmetric tetrabranched PEG or an asymmetric bibranched PEG.

[0013] According to certain embodiments, the molecular weight of each branch of the asymmetric branched PEG is 500-5000 Daltons, for example, 500 Daltons, 1000 Daltons, 1500 Daltons, 2000 Daltons, 2500 Daltons, 3000 Daltons, 3500 Daltons, 4000 Daltons, 4500 Daltons, 5000 Daltons or any value between them.

[0014] According to certain implementations, the asymmetric branched PEG can be an asymmetric bibranched PEG.

[0015] According to certain implementations, the polyamino organic acid connector may be a diamino organic acid connector.

[0016] According to some embodiments, the polyamino organic acid linker may be a lysine linker.

[0017] According to certain preferred embodiments, the asymmetric branched PEG-modified lipid compound may be a compound of formula (I) as follows:

[0018] Where m is 12-124; and n is 12-124, and m is not equal to n.

[0019] In some implementations, n is 37-124.

[0020] In some implementations, m is selected from 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124 or any value between them.

[0021] In some implementations, n is selected from 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 37, 38, 40, 42, 44, 46, 48, 49, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124 or any value between them.

[0022] In some embodiments, the difference between m and n is at least 1. In some embodiments, the difference between m and n is 1-112, for example, 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 37, 38, 40, 42, 44, 46, 48, 49, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, or any value or range thereof. In some preferred embodiments, the difference between m and n is 25-100, or any value between thereof.

[0023] In some implementations, m = 24 and n = 49.

[0024] In some implementations, m = 12 and n = 37.

[0025] In some implementations, m = 24 and n = 124.

[0026] In some implementations, m = 124 and n = 49.

[0027] In some embodiments, the asymmetric branched PEG-modified lipid compound is selected from:

[0028] The molecular weight of PEG with 12 units is 500 Daltons, i.e., PEG500; the molecular weight of PEG with 24 units is 1000 Daltons, i.e., PEG1000 (PEG1k); the molecular weight of PEG with 37 units is 1500 Daltons, i.e., PEG1500 (PEG1.5k); the molecular weight of PEG with 49 units is 2000 Daltons, i.e., PEG2000 (PEG2k); and the molecular weight of PEG with 124 units is 5000 Daltons, i.e., PEG5000 (PEG5k). Therefore, the molecular weight of each branch of the asymmetric branched PEG described in this invention is 500-5000 Daltons.

[0029] A second aspect of the present invention provides a method for preparing the asymmetric branched PEG-modified compound of formula (I) disclosed herein, comprising the following steps:

[0030] 1) Synthesize compound C from compound A and compound B.

[0031] 2) Synthesize compound D from compound C.

[0032] 3) Synthesize compound E from compound D.

[0033] 4) Synthesize compound G from compound E and compound F.

[0034] 5) Synthesize compound H from compound G

[0035] 6) Synthesize compound J from compound I

[0036] as well as

[0037] 7) Synthesize compound (I) from compound H and compound J.

[0038] In step 3), during the synthesis of compound E from compound D, triphosgene refers to triphosgene, whose full chemical name is bis(trichloromethyl) carbonate.

[0039] A third aspect of the invention provides the use of the asymmetric branched PEG-modified lipid compounds of this disclosure in the preparation of nucleic acid delivery systems.

[0040] According to certain implementations, the nucleic acid may be selected from DNA, messenger RNA (mRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribosomal RNA (rRNA), transport RNA (tRNA), circular RNA (circRNA), aptamers, or plasmids.

[0041] According to some implementation schemes, the nucleic acid may be mRNA.

[0042] According to some implementation schemes, the nucleic acid encodes a therapeutic protein.

[0043] According to certain implementations, the nucleic acid delivery system may be an LNP.

[0044] A fourth aspect of the present invention provides a nucleic acid delivery system comprising one or more of the following: an asymmetric branched PEG-modified lipid compound of the present disclosure, a cationic / ionizable lipid, an auxiliary lipid, cholesterol, and a nucleic acid.

[0045] According to certain embodiments, the cationic / ionizable lipids include, but are not limited to, one or more combinations of SM102, Dlin-KC2-DMA, Dlin-MC3-DMA, DOTAP, or ALC-0315.

[0046] According to certain embodiments, the auxiliary lipids include, but are not limited to, one or more combinations of distearylphosphatidylcholine (DSPC) or a derivative thereof, distearylphosphatidylethanolamine (DSPE) or a derivative thereof, stearoyloleoylphosphatidylcholine (SOPC) or a derivative thereof, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE) or a derivative thereof, N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP) or a derivative thereof, and 1,2-dipalmitoyl-sn-glycerol-3-o-4'-(N,N,N-trimethyl)homoserine (DGTS) or a derivative thereof.

[0047] According to certain implementation schemes, the nucleic acid may be selected from one or more combinations of DNA, messenger RNA (mRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribosomal RNA (rRNA), transport RNA (tRNA), circular RNA (circRNA), aptamers, or plasmids.

[0048] According to some implementation schemes, the nucleic acid may be mRNA.

[0049] According to some implementation schemes, the nucleic acid encodes a therapeutic protein.

[0050] According to certain implementations, the nucleic acid delivery system may be an LNP.

[0051] The asymmetric branched PEG-modified lipid compound and the nucleic acid delivery system containing it described in this invention have the characteristics of inducing low levels of anti-PEG IgM and anti-PEG IgG in vivo after repeated injections, and high in vivo transfection efficiency.

[0052] Compared with the prior art, the asymmetric branched PEG-modified lipid compound and the nucleic acid delivery system containing it developed in this invention have at least the following beneficial effects:

[0053] 1. The asymmetric branched PEG-modified lipid compounds of the present invention are used in nucleic acid delivery systems, especially mRNA-LNP delivery systems, which produce low levels of anti-PEG IgM and anti-PEG IgG in subjects after repeated injections.

[0054] 2. The asymmetric branched PEG-modified lipid compounds of the present invention are used in nucleic acid delivery systems, especially mRNA-LNP delivery systems, which have high transfection efficiency in subjects after repeated injections.

[0055] 3. The lipid nanoparticles prepared from the asymmetric branched PEG-modified lipid compounds of the present invention have small particle size and good uniformity, and their encapsulation efficiency can reach up to 91.22%. Attached Figure Description

[0056] Figure 1 is a conceptual schematic diagram of asymmetric branched PEG-modified lipid molecules prepared according to Examples 1, 2, 3 and 4 of the present invention, where x and y represent the molecular weight of PEG.

[0057] Figure 2a shows the level of anti-PEG IgG induced in BALB / c mice after injection of asymmetric branched PEG-modified lipid nanoparticles prepared according to Example 5 of the present invention and control lipid nanoparticles, indicating in vivo immunogenicity.

[0058] Figure 2b shows the level of anti-PEG IgM induced in BALB / c mice after injection of asymmetric branched PEG-modified lipid nanoparticles prepared according to Example 5 of the present invention and control lipid nanoparticles, indicating in vivo immunogenicity.

[0059] Figure 3 shows the fluorescence intensity of the asymmetric branched PEG-modified lipid nanoparticles prepared according to Example 5 of the present invention and the control lipid nanoparticles after intramuscular injection in mice, indicating the in vivo delivery / transfection efficiency. Detailed Implementation

[0060] To further illustrate the technical means and effects adopted by the present invention to achieve the intended purpose, the specific implementation methods, structures, features and effects according to the present invention will be described in detail below with reference to the accompanying drawings and preferred embodiments.

[0061] Example 1: Preparation of asymmetric branched PEG-modified lipid compound Chol-PEG1k / 2k (of formula (I) where m=24 and n=49)

[0062] The specific structure of compound Chol-PEG1k / 2k is as follows:

[0063] Compound Chol-PEG1k / 2k

[0064] The preparation steps are as follows:

[0065] 1) Synthesis of compound C

[0066] EDCI (4.96 g, 25.8 mmol) and DMAP (316 mg, 2.59 mmol) were added to a DCM (30.0 mL) solution containing compound A (5.00 g, 12.9 mmol) and compound B (7.27 g, 15.52 mmol). The mixture was stirred at 25 °C for 12 h. TLC (dichloromethane DCM:methanol MeOH = 10:1) showed a small residue of compound A and the formation of a new principal spot. The reaction was terminated by adding 100 mL of H2O. The mixture was extracted with ethyl acetate (100 mL x 3), and the combined organic layers were washed with brine (100 mL), dried with Na2SO4, filtered, and concentrated under reduced pressure to give the crude product. The crude product was then analyzed by rapid silica gel chromatography (…). 40g Purification was performed using a silica gel rapid chromatography column (elution buffer: 0-2% methanol / dichloromethane, 40 mL / min) to give a yellow solid compound C (6.9 g, 8.24 mmol, yield 63.7%).

[0067] 2) Synthesis of compound D

[0068] DBU (4.00 g, 26.2 mmol, 3.96 mL) was added to a DCM solution (5.50 g, 6.57 mmol) containing compound C (3.50 mL). The mixture was stirred at 25 °C for 2.5 h. TLC (dichloromethane DCM:methanol MeOH = 10:1) showed that compound C was completely consumed. The reaction mixture was concentrated under reduced pressure to give the crude product, which was then analyzed by rapid silica gel chromatography (…). 20g Purification was performed using a silica gel rapid chromatography column with 0-5% methanol / dichloromethane as eluent at 30 mL / min to give yellow oily compound D (3.6 g, 5.85 mmol, yield 89.11%).

[0069] 3) Synthesis of compound E

[0070] At 0 °C, bis(trichloromethyl) carbonate (830 mg, 2.80 mmol) was added to a THF (5.00 mL) solution containing compound D (2.26 g, 3.68 mmol) and TEA (743 mg, 7.35 mmol, 1.02 mL), and the mixture was stirred at 60 °C for 2 h. TLC (petroleum ether PE: ethyl acetate EA = 0:1) showed that compound D was completely consumed. The reaction mixture was concentrated under vacuum to give a yellow solid compound E (2.36 g, 3.68 mmol).

[0071] 4) Synthesis of compound G

[0072] A mixture of compound E (2.36 g, 3.68 mmol), compound F (2.06 g, 920 μmol), and TEA (745 mg, 7.36 mmol, 1.03 mL) was dissolved in toluene (20.0 mL) at 25 °C, degassed, and purged three times with N2. The mixture was then stirred at 100 °C for 16 h under N2 protection. TLC (dichloromethane DCM:methanol MeOH = 10:1) showed a trace residue of compound F. The reaction mixture was concentrated under vacuum to obtain the residue. The residue was analyzed by rapid silica gel chromatography (…). 12g Purification was performed using a silica gel rapid chromatography column (elution buffer: 0-5% methanol / dichloromethane, 30 mL / min) to give a yellow solid compound G (920 mg, 312 μmol, yield 8.50%, purity 97.8%).

[0073] 5) Synthesis of compound H

[0074] At 25°C, HCl / EtOAc (2M, 10.0mL) was added to a DCM solution (900mg, 312μmol) containing compound G (10.0mL), and the mixture was stirred for 1 h. TLC (dichloroethane DCM:methanol MeOH = 10:1) showed that compound G was completely consumed. The reaction mixture was concentrated under vacuum to give a yellow solid compound H (900mg, crude product).

[0075] 6) Synthesis of compound J

[0076] At 25 °C, (4-nitrophenyl)chloroformate (2.67 g, 13.2 mmol) was added to a DCM solution (50 mL) containing compound I (5.00 g, 4.41 mmol) and TEA (1.79 g, 17.6 mmol, 2.46 mL), and the mixture was stirred at 25 °C for 48 h. TLC (dichloromethane DCM:methanol MeOH = 10:1) showed that compound I remained. The reaction mixture was washed with an aqueous solution of Na₂CO₃ (50 mL x 10), dried over Na₂SO₄, filtered, and concentrated under reduced pressure to give a yellow solid compound J (4.20 g, 3.23 mmol, yield 73.3%).

[0077] 7) Synthesis of compound Chol-PEG1k / 2k

[0078] A mixture of compound H (1.1 g, 396 μmol), compound J (1.03 g, 792 μmol), and TEA (160 mg, 1.59 mmol) was dissolved in DCM (10.0 mL) at 0 °C, degassed, and purged three times with N2. The mixture was stirred at 25 °C for 13 h under N2 conditions. TLC (DCM: dichloromethane: MeOH / methanol = 10:1) showed that compound H was completely consumed. The reaction mixture was concentrated under vacuum to obtain the residue, which was then subjected to rapid silica gel chromatography (…). 12g The crude product was purified by a silica gel rapid chromatography column (elution buffer: 0-5% methanol / dichloromethane, 30 mL / min). The crude product was then purified by HPLC (column: Welch Xltimate C4 100*30*10 μm; mobile phase: [water (trifluoroacetic acid)-methanol]; gradient: 30%-90% B, 22 min) to obtain a white solid compound, Chol-PEG1k / 2k (500 mg, 126 μmol, yield 32.0%, purity 99.87%).

[0079] The NMR data for compound Chol-PEG1k / 2k are as follows: 1 H NMR (400MHz CDCl3)

[0080] δ5.36-5.49(m,2H),4.94-5.04(m,1H),4.60-4.73(m,1H),4.16-4.32(m,5H),3.80-3.86(m ,2H),3.59-3.74(m,281H),3.54-3.57(m,4H),3.45-3.50(m,2H),3.38(s,6H),3.12-3.21(m ,2H),2.29-2.38(m,2H),1.92-2.03(m,2H),1.78-1.91(m,4H),1.44-1.73(m,10H),1.30-1 .42(m,5H),1.06-1.21(m,7H),0.90-1.03(m,9H),0.87(dd,J=6.4,1.6Hz,6H),0.68(s,3H).

[0081] Example 2: Preparation of asymmetric branched PEG-modified lipid compound Chol-PEG500 / 1500 (of formula (I) where m=12 and n=37)

[0082] The specific structure of compound Chol-PEG500 / 1500 is as follows:

[0083] Compound Chol-PEG500 / 1500

[0084] The preparation steps are as follows:

[0085] 1) Repeat steps 1) to 3) of Example 1 to prepare compound E.

[0086] 2) Synthesis of compound L

[0087] A mixture of compound E (10.0 g, 15.6 mmol), compound K (6.65 g, 3.90 mmol), and TEA (3.16 g, 31.2 mmol, 4.34 mL) was dissolved in toluene (150 mL) at 25 °C, degassed, and purged three times with N2. The mixture was then stirred at 100 °C for 16 h under N2 protection. TLC (dichloromethane DCM:methanol MeOH = 10:1) showed a trace residue of compound K. The reaction mixture was concentrated under vacuum to obtain the residue. The residue was analyzed by rapid silica gel chromatography (…). 40g Purification was performed using a silica gel rapid chromatography column (elution buffer: 0-5% methanol / dichloromethane, 50 mL / min) to give a yellow solid compound L (4.8 g, 2.05 mmol, yield 13.11%).

[0088] 3) Synthesis of compound M

[0089] At 25°C, HCl / EtOAc (4M, 25.0mL) was added to a DCM solution (50.0mL) containing compound L (4.8mg, 2.05mmol), and the mixture was stirred for 30 min. TLC (dichloroethane DCM:methanol MeOH = 10:1) showed that compound L was completely consumed. The reaction mixture was concentrated under vacuum to give a yellow solid compound M (4.4g, crude product).

[0090] 4) Synthesis of compound O

[0091] At 0 °C, (4-nitrophenyl)chloroformate (4.00 g, 19.8 mmol) was added to a DCM solution (50 mL) containing compound N (3.00 g, 4.96 mmol) and TEA (3.01 g, 29.7 mmol, 4.14 mL), and the mixture was stirred at 25 °C for 12 h. TLC (dichloromethane DCM:methanol MeOH = 10:1) showed the residue of compound N. The mixture was diluted with DCM (100 mL) and the reaction mixture was washed with Na2CO3 aqueous solution (50 mL x 10), dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was subjected to rapid silica gel chromatography (…). 20g Purification was performed using a silica gel rapid chromatography column (eluting with 0-5% methanol / dichloromethane at 30 mL / min) to give a yellow solid compound O (3.30 g, 4.29 mmol, yield 86.4%).

[0092] 5) Synthetic compound Chol-PEG500 / 1500

[0093] A mixture of compound M (4.4 g, 1.96 mmol), compound O (1.51 g, 1.96 mmol), and TEA (792 mg, 7.83 mmol, 1.09 mL) was dissolved in DCM (40.0 mL) at 0 °C, degassed, and purged three times with N2. The mixture was stirred at 25 °C for 13 h under N2 conditions. TLC (DCM: dichloromethane: MeOH / methanol = 10:1) showed that compound M was completely consumed. The reaction mixture was concentrated under vacuum to obtain the residue, which was then subjected to rapid silica gel chromatography (…). 12g The compound Chol-PEG500 / 1500 (510 mg, 172.98 μmol, yield 8.83%, purity 97.6%) was purified by silica gel rapid chromatography column (elution buffer: 0-5% methanol / dichloromethane, 30 mL / min) to obtain a white solid compound.

[0094] The NMR data for compound Chol-PEG500 / 1500 are as follows: 1 H NMR (400MHz CDCl3)

[0095] δ5.34-5.44(m,2H),4.88-4.99(m,1H),4.58-4.71(m,1H),4.15-4.31(m,5H),3.78-3.85(m,1H ),3.56-3.77(m,174H),3.52-3.55(m,4H),3.42-3.49(m,1H),3.31-3.41(m,6H),3.10-3.18(m ,2H),2.24-2.36(m,2H),1.92-2.03(m,2H),1.76-1.89(m,4H),1.43-1.67(m,10H),1.29-1.40 (m,5H),1.06-1.21(m,7H),0.89-1.03(m,9H),0.85(dd,J=6.4,1.6Hz,6H),0.63-0.69(m,3H).

[0096] Example 3: Preparation of asymmetric branched PEG-modified lipid compound Chol-PEG1k / 5k (compound of formula (I) where m=24 and n=124)

[0097] The specific structure of compound Chol-PEG1k / 5k is as follows:

[0098] Compound Chol-PEG1k / 5k

[0099] The preparation steps are as follows:

[0100] 1) Repeat steps 1) to 3) of Example 1 to prepare compound E.

[0101] 2) Synthesis of compound Q

[0102] A mixture of compound E (1.00 g, 1.56 mmol), compound P (2.16 g, 390 μmol), and TEA (316 mg, 3.12 mmol) was dissolved in toluene (30.0 mL) at 25 °C, degassed, and purged three times with N2. The mixture was then stirred at 100 °C for 16 h under N2 protection. TLC (dichloromethane DCM:methanol MeOH = 10:1) showed a small residue of compound P. The reaction mixture was concentrated under vacuum to obtain the residue. The residue was analyzed by rapid silica gel chromatography (…). 40g Purification was performed using a silica gel rapid chromatography column (elution buffer: 0-5% methanol / dichloromethane, 40 mL / min) to give a yellow solid compound Q (1.4 g, 227 μmol, yield 14.52%).

[0103] 3) Synthesis of compound R

[0104] At 25°C, HCl / EtOAc (2M, 14.0mL) was added to a DCM solution (1.4g, 226μmol) containing compound Q (1.4g). The mixture was stirred for 12h. TLC (dichloroethane DCM:methanol MeOH = 10:1) showed that compound Q was completely consumed. The reaction mixture was concentrated under vacuum to give a yellow solid compound R (1.4g, crude product).

[0105] 4) Repeat step 6) in Example 1 to synthesize compound J

[0106] 5) Synthesize compound Chol-PEG1k / 5k

[0107] A mixture of compound R (1.4 g, 230 μmol), compound J (360 mg, 277 μmol), and TEA (93.2 mg, 921 μmol) was dissolved in DCM (10.0 mL) at 0 °C, degassed, and purged three times with N2. The mixture was stirred at 25 °C for 13 h under N2 conditions. TLC (DCM: dichloromethane: MeOH / methanol = 10:1) showed that compound R was completely consumed. The reaction mixture was concentrated under vacuum to obtain the residue, which was then subjected to rapid silica gel chromatography (…). 12g The crude product was purified using a silica gel rapid chromatography column (eluting with 0-5% methanol / dichloromethane, 30 mL / min). The crude product was then purified to give a white solid compound, Chol-PEG1k / 5k (360 mg, 48.9 μmol, yield 21.25%, purity 98.42%).

[0108] The NMR data for compound Chol-PEG1k / 5k are as follows: 1 H NMR (400MHz CDCl3)

[0109] δ5.36-5.49(m,2H),4.94-5.04(m,1H),4.60-4.73(m,1H),4.16-4.35(m,5H),3.80-3.86(m, 2H),3.59-3.74(m,563H),3.54-3.57(m,8H),3.45-3.50(m,3H),3.37(s,6H),3.12-3.21(m,2 H),2.25-2.35(m,2H),1.92–1.97(m,2H),1.78-1.90(m,4H),1.44-1.72(m,10H),1.24-1.36( m,5H),1.05-1.21(m,7H),0.89-1.04(m,9H),0.86(dd,J=6.4,1.6Hz,6H),0.63-6.70(m,3H).

[0110] Example 4: Preparation of asymmetric branched PEG-modified lipid compound Chol-PEG2k / 5k (compound of formula (I) where m=124 and n=49)

[0111] The specific structure of compound Chol-PEG2k / 5k is as follows:

[0112] Compound Chol-PEG2k / 5k

[0113] The preparation steps are as follows:

[0114] 1) Repeat steps 1) to 5) of Example 1 to obtain compound H.

[0115] 2) Synthesis of compound S

[0116] At 25 °C, (4-nitrophenyl)chloroformate (545 mg, 2.71 mmol) was added to a DCM solution (50 mL) containing compound P (5.00 g, 920 μmol) and TEA (365 mg, 3.61 mmol), and the mixture was stirred at 25 °C for 48 h. TLC (dichloromethane DCM:methanol MeOH = 10:1) showed that compound S remained. The reaction mixture was washed with an aqueous solution of Na₂CO₃ (50 mL x 10), dried over Na₂SO₄, filtered, and concentrated under reduced pressure to give compound S (4.2 g, 736 μmol, yield 81.57%) as a yellow solid.

[0117] 3) Synthesis of compound Chol-PEG2k / 5k

[0118] A mixture of compound H (1.0 g, 360 μmol), compound S (2.47 g, 432 μmol), TEA (109 mg, 1.08 mmol, 1.09 mL), and DMAP (8.80 mg, 72.06 μmol) was dissolved in DCM (15.0 mL) at 0 °C, and the mixture was stirred at 25 °C for 2 h. TLC (DCM: dichloromethane: MeOH / methanol = 10:1) showed that compound H was completely consumed. The reaction mixture was concentrated under vacuum to obtain a residue, which was then subjected to rapid silica gel chromatography (…). 12g The crude product was purified by a silica gel rapid chromatography column (elution buffer: 0-5% methanol / dichloromethane, 30 mL / min). The crude product was then purified by HPLC (column: Welch Xltimate C4 100*30*10 μm; mobile phase: [water (trifluoroacetic acid)-methanol]; gradient: 30%-92% B, 20.2 min) to obtain a white solid compound, Chol-PEG2k / 5k (280 mg, 33.2 μmol, yield 9.23%, purity 99.02%).

[0119] The NMR data for compound Chol-PEG2k / 5k are as follows: 1 H NMR (400MHz CD3OD)

[0120] δ5.38-5.43(m,1H),4.52-4.61(m,1H),4.04-4.23(m,3H),3.79-3.85(m,2 H),3.52-3.72(m,690H),3.45-3.48(m,4H),3.36(s,6H),3.07-3.13(m,3H) ,2.30-2.39(m,2H),1.76-2.10(m,6H),1.46-1.75(m,10H),1.35-1.45(m, 5H),1.09-1.25(m,7H),0.92-1.07(m,9H),0.87-0.91(m,6H),0.73(s,3H).

[0121] The materials used in the embodiments of this application are from the following sources:

[0122] The following examples illustrate the technical effects of applying the PEG lipid compounds Chol-PEG1k / 2k, Chol-PEG500 / 1500, Chol-PEG1k / 5k, and Chol-PEG2k / 5k prepared in Examples 1, 2, 3, and 4, respectively, to an mRNA-LNP delivery system.

[0123] Example 5: Preparation and detection of lipid nanoparticles containing Chol-PEG1k / 2k, Chol-PEG500 / 1500, Chol-PEG1k / 5k, or Chol-PEG2k / 5k

[0124] 1) Preparation steps

[0125] The PEG lipid compounds Chol-PEG1k / 2k, Chol-PEG500 / 1500, Chol-PEG1k / 5k, and Chol-PEG2k / 5k prepared in Examples 1, 2, 3, and 4 respectively were dissolved in ethanol at a molar ratio of 1.0:50:39:10 to prepare ethanol lipid solutions. Firefly luciferase (Fluc) mRNA was diluted with 10 mM acetate buffer (pH = 4.0) to obtain an aqueous solution of Fluc mRNA. The ethanol lipid solution and the above-mentioned mRNA aqueous solution were mixed at a volume ratio of 1:3 using a microfluidic device to prepare lipid nanoparticles. These nanoparticles were then dissolved in 40 times the volume of the lipid nanoparticle solution in Tris (tris(hydroxymethyl)aminomethane) pH 7.5 solution, stirred for at least 5 minutes, and then subjected to ultrafiltration to remove ethanol before concentrating the sample. Finally, the concentrated sample solution was sterile filtered (0.2 μm) to obtain lipid nanoparticles encapsulating firefly luciferase (Fluc) mRNA, which were named Chol-PEG1k / 2k lipid nanoparticles, Chol-PEG500 / 1500 lipid nanoparticles, Chol-PEG1k / 5k lipid nanoparticles, and Chol-PEG2k / 5k lipid nanoparticles, respectively, and abbreviated as Chol-PEG1k / 2k LNP, Chol-PEG500 / 1500 LNP, Chol-PEG1k / 5k LNP, and Chol-PEG2k / 5k LNP, respectively.

[0126] In addition, lipid nanoparticles containing the compound DMG-PEG2k were prepared using the same method as described above as a control, and named as DMG-PEG2k lipid nanoparticles, or simply DMG-PEG2k LNP.

[0127] DMG-PEG2k (dimyristicoyl-sn-glycerol-polyethylene glycol 2000) is the PEG lipid used in Moderna's COVID-19 vaccine, and its structure is as follows:

[0128] The molar ratio of DMG-PEG2k, SM102, cholesterol and DSPC is 1.5:50:38.5:10.

[0129] 2) Testing steps

[0130] The particle size and polydispersity index (PDI) of the nanoparticles were determined using a Malvern particle size analyzer. Particle size was measured in Tris solution at pH 7.5. The encapsulation efficiency (EE%) of the lipid nanoparticles was determined by ion exchange chromatography. The test results are shown in Table 1 below:

[0131] Table 1:

[0132] As shown in Table 1, the lipid nanoparticles prepared using the lipid compounds Chol-PEG1k / 2k, Chol-PEG500 / 1500, Chol-PEG1k / 5k, and Chol-PEG2k / 5k prepared in Examples 1, 2, 3, and 4 all have small particle size and good uniformity, with encapsulation efficiencies all exceeding 80%, reaching a maximum of 91.22%, roughly equivalent to the performance of DMG-PEG2k lipid nanoparticles. This indicates that the Chol-PEG1k / 2k, Chol-PEG500 / 1500, Chol-PEG1k / 5k, and Chol-PEG2k / 5k lipid compounds prepared in Examples 1, 2, 3, and 4 can form lipid nanoparticles as PEG lipids.

[0133] Example 6: Detection experiment of anti-PEG binding antibodies (anti-PEG IgG binding antibody and anti-PEG IgM binding antibody) on the lipid nanoparticles prepared in Example 5.

[0134] 1) Detection of specific anti-PEG IgG binding antibodies

[0135] The level of specific anti-PEG IgG antibodies in the plasma of immunized animals was detected by indirect ELISA. Serum samples were diluted and incubated in pre-coated PEG antigen-containing plates for 1 h, followed by washing with PBST. HRP-conjugated antibody (anti-mouse IgG HRP) was then added and incubated at room temperature for 45 min, followed by washing with PBST. Color development was achieved by incubation with TMB solution at room temperature for 20 min, and the reaction was terminated by adding stop solution. The absorbance was measured at 450 nm to determine the level of anti-PEG IgG antibodies.

[0136] The results are shown in Figure 2a (Figure: leftmost column is DMG-PEG2k, middle column is Chol-PEG1k / 2k, rightmost column is Chol-PEG2k / 5k). Mice were first injected with LNP on day 0 and a second injection was given on day 7 (7D). Testing was performed on day 14 (14D). The levels of anti-PEG IgG induced in vivo by Chol-PEG1k / 2k and Chol-PEG2k / 5k lipid nanoparticles were significantly lower than those induced in vivo by the control DMG-PEG2k lipid nanoparticles. This indicates that the lipid nanoparticles prepared using the asymmetric branched PEG-modified lipid compounds of this invention induce significantly lower levels of anti-PEG IgG in vivo under repeated injections compared to the control, suggesting lower in vivo immunogenicity.

[0137] 2) Detection of specific anti-PEG IgM binding antibodies

[0138] The level of specific anti-PEG IgM antibody in the plasma of immunized animals was detected by indirect ELISA. Serum samples were diluted and incubated in pre-coated PEG antigen-containing plates for 1 h, followed by washing with PBST. HRP-conjugated antibody (anti-mouse IgM HRP) was then added and incubated at room temperature for 45 min, followed by washing with PBST. TMB was used for color development, and the reaction was incubated at room temperature for 20 min. The reaction was terminated by adding stop solution, and the absorbance was measured at 450 nm to determine the level of anti-PEG IgM antibody. The results are shown in Figure 2b (Figure: the leftmost column is DMG-PEG2k, the second column from the left is Chol-PEG1k / 2k, the third column from the left is Chol-PEG1k / 5k, and the rightmost column is Chol-PEG2k / 5k).

[0139] Mice were first injected with LNP on day 0 and a second injection was given on day 7 (7D). Testing was performed on day 14 (14D). The anti-PEG IgM levels induced in vivo by Chol-PEG1k / 2k, Chol-PEG1k / 5k, and Chol-PEG2k / 5k lipid nanoparticles were significantly lower than those induced in vivo by the control DMG-PEG2k lipid nanoparticles. This indicates that the lipid nanoparticles prepared using the asymmetric branched PEG-modified lipid compounds of this invention induce significantly lower anti-PEG IgM levels in vivo under repeated injections compared to the control, suggesting lower in vivo immunogenicity.

[0140] Example 7: In vivo transfection experiment of lipid nanoparticles prepared in Example 5

[0141] Three groups of BALB / c mice (n=3 per group) were intramuscularly injected once on day 0 and day 7 with control DMG-PEG2k lipid nanoparticles, Chol-PEG1k / 2k lipid nanoparticles, or Chol-PEG500 / 1500 lipid nanoparticles (3 mg / kg each time). Immediately after the second injection, a luciferase substrate was injected intraperitoneally. Fluorescence intensity was measured 4 hours after the second injection using a small animal in vivo imaging system. Higher fluorescence intensity indicated higher LNP delivery / transfection efficiency.

[0142] The fluorescence intensity results are shown in Figure 3. The fluorescence intensity of the Chol-PEG1k / 2k lipid nanoparticles and Chol-PEG500 / 1500 lipid nanoparticles of the present invention was higher than that of the control group 4 hours after the second injection.

[0143] As can be seen, liposome nanoparticles prepared using the asymmetric branched PEG-modified lipid compounds of the present invention achieve higher in vivo delivery / transfection efficiency under repeated injection, consistent with the lower levels of anti-PEG antibodies induced in vivo under repeated injection demonstrated in Example 6. Without being limited by theory, it is believed that this higher in vivo delivery / transfection efficiency is precisely due to the lower levels of anti-PEG antibodies induced in vivo.

[0144] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention shall still fall within the scope of the technical solution of the present invention.

[0145] References:

[0146] 1.Ju Y, Lee WS et al. Anti-PEG antibodies boosted in humans by SARS-Cov-2lipid nanoparticle mRNA vaccine. ACS Nano, 2023; 16:11769-11780.

[0147] 2. GT Kozma, T. Meszaros, P. Berenyi et al. Role of anti-polyethylene glycol (PEG)antibodies in the allergic reactions to PEG-containing Covid-19 vaccines: Evidence for immunogenicity of PEG. Vaccine, 2023; 41: 4561-4570.

[0148] 3. Ibrahim M, Ramadan E et al. Polyethylene glycol (PEG): The nature, immunogenicity, and role in the hypersensitivity of PEGylated products. Journal of controlled Release, 2022; 351:215-230.

[0149] 4. Chien CS et al. Using cationic polyurethane-short branch PEI as microRNA-driven nano-delivery system for stem cell differentiation. Chinese Medical Association, 2020; 83(4):367-370.

[0150] 5. Park Y et al. Poly(aspartic acid)-based polymeric nanoparticle for local and systemic mRNA delivery. Molecular Pharmaceutics, 2022; 19(12): 4696–4704.

[0151] 6. Abu Lila AS et al. Application of polyglycerol coating to plasmid DNA lipoplex for the evasion of the accelerated blood clearance phenomenon in nucleic acid delivery.Journal of Pharmaceutical Sciences, 2014; 103(2):557-66.

[0152] 7. Zeng H et al. Amino Acid-Functionalized Dendritic Polyglycerol for Safe and Effective siRNA Delivery. Biomacromolecules, 2015; 16(12):3869-77.

[0153] 8. Soliman OY et al. Efficiency of Chitosan / Hyaluronan-Based mRNA Delivery Systems In Vitro: Influence of Composition and Structure. Journal of Pharmaceutical Sciences, 2020; 109 (4): 1581 - 1593.

[0154] 9. Duan X, Zhang Y, Guo M et al. Sodium alginate coating simultaneously increases the biosafety and immunotherapeutic activity of the cationic mRNA nanovaccine. Acta Pharmaceutica Sinica B, 2023, 13 (3): 942-954.

Claims

1. An asymmetric branched PEG-modified lipid compound having the following structural formula: Asymmetric branched PEG-polyamino organic acid linker-cholesterol.

2. The lipid compound modified with asymmetric branched PEG according to claim 1, wherein the asymmetric branched PEG is an asymmetric tetrabranched PEG or an asymmetric dibranched PEG; Preferably, the molecular weight of each branch of the asymmetric branched PEG is 500-5000 Daltons.

3. The lipid compound modified with asymmetric branched PEG according to claim 2, wherein the asymmetric branched PEG is an asymmetric bibranched PEG.

4. The asymmetric branched PEG-modified lipid compound according to claim 1, wherein the polyamino organic acid linker is a diamino organic acid linker.

5. The asymmetric branched PEG-modified lipid compound according to claim 1, wherein the polyamino organic acid linker is a lysine linker.

6. The asymmetric branched PEG-modified lipid compound according to claim 1, wherein it is a compound of formula (I) as follows: Where m is 12-124; and n is 12-124, and m is not equal to n.

7. The asymmetric branched PEG-modified lipid compound according to claim 6, wherein n is 37-124.

8. The asymmetric branched PEG-modified lipid compound according to claim 6, wherein m is selected from 12, 24 and 124.

9. The asymmetric branched PEG-modified lipid compound according to claim 6, wherein n is selected from 12, 37, 49 and 124.

10. The asymmetric branched PEG-modified lipid compound according to claim 6, wherein m = 24 and n = 49.

11. The asymmetric branched PEG-modified lipid compound according to claim 6, wherein m = 12 and n = 37.

12. The asymmetric branched PEG-modified lipid compound according to claim 6, wherein m = 24 and n = 124.

13. The asymmetric branched PEG-modified lipid compound according to claim 6, wherein m = 124 and n = 49.

14. The asymmetric branched PEG-modified lipid compound according to claim 6, wherein the asymmetric branched PEG-modified lipid compound is selected from:

15. A method for preparing the asymmetric branched PEG-modified lipid compound according to any one of claims 6-14, comprising the following steps: 1) Synthesis of compound C from compound A and compound B 2) synthesis of compound D from compound C 3) Synthesize compound E from compound D. 4) synthesis of compound G from compound E and compound F 5) Synthesis of compound H from compound G 6) synthesis of compound J from compound I as well as 7) Synthesize compound (I) from compound H and compound J.

16. Use of the asymmetric branched PEG-modified lipid compound according to any one of claims 1-14 in the preparation of a nucleic acid delivery system.

17. The use according to claim 16, wherein the nucleic acid is selected from DNA, messenger RNA (mRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribosomal RNA (rRNA), transport RNA (tRNA), circular RNA (circRNA), aptamers, or plasmids.

18. The use according to claim 17, wherein the nucleic acid is mRNA.

19. The use according to claim 16, wherein the nucleic acid delivery system is an LNP.

20. The use according to claim 17, wherein the nucleic acid encodes a therapeutic protein.

21. A nucleic acid delivery system comprising one or more of the following: an asymmetric branched PEG-modified lipid compound according to any one of claims 1-14, a cationic / ionizable lipid, an auxiliary lipid, cholesterol, and a nucleic acid.

22. The system of claim 21, wherein the nucleic acid is selected from DNA, messenger RNA (mRNA), small interfering RNA (siRNA), microRNA (miRNA), short hairpin RNA (shRNA), ribosomal RNA (rRNA), transport RNA (tRNA), circular RNA (circRNA), aptamers, or plasmids.

23. The system of claim 22, wherein the nucleic acid is mRNA.

24. The system of claim 21, wherein the nucleic acid delivery system is an LNP.

25. The system of claim 21, wherein the nucleic acid encodes a therapeutic protein.

Images