A lipid compound, a preparation method and application thereof, and a pharmaceutical composition

Abstract

This invention provides a lipid compound, its preparation method and application, and a pharmaceutical composition, belonging to the pharmaceutical field. Through innovative design of the chemical structure of lipid molecules, particularly by specific modification of the hydrophilic head group and / or hydrophobic tail structure, this invention enables specific expression of mRNA in the liver, effectively avoiding off-target effects in non-target organs, and has significant scientific value and broad application prospects.

Description

Technical Field

[0001] This invention relates to the pharmaceutical field, specifically to a lipid compound, its preparation method and application, and pharmaceutical compositions. Background Technology

[0002] In recent years, advancements in molecular biology techniques have significantly propelled the innovative development of mRNA drugs, demonstrating revolutionary potential, particularly in the fields of vaccines and protein replacement therapies. The core advantage of mRNA drugs lies in their ability to rapidly encode specific functional proteins, drastically shortening drug development cycles and providing a platform for personalized treatment. Compared to traditional drugs, mRNA drugs do not require the direct introduction of antigens or drug molecules; instead, they guide the body to synthesize target proteins by delivering genetic instructions, thus offering unique advantages in addressing emerging infectious diseases, cancer, and genetic disorders. However, the clinical application of mRNA molecules faces the dual challenges of delivery efficiency and stability. mRNA is easily degraded by nucleases in vivo and struggles to autonomously penetrate cell membrane barriers; therefore, its efficacy is highly dependent on the design of the delivery system.

[0003] Lipid nanoparticles (LNPs), as a cutting-edge nucleic acid delivery technology, offer a possibility for achieving this goal. A typical LNP consists of four core components: ionizable lipids, accessory lipids, structural lipids, and polyethylene glycol-modified (PEG) lipids. Among these, the molecular structure of the ionizable lipids is crucial in determining the biological fate of the LNP. It is not only responsible for protonation in acidic endosomes to achieve intracellular drug release, but more importantly, it profoundly influences the tissue distribution of the LNP in vivo.

[0004] The structural characteristics of lipids, such as their head groups, linkages, length, saturation, and branching, collectively determine the particle size, surface charge, flexibility, and affinity for plasma proteins like ApoE. Subtle differences in these properties can lead to vastly different targeting capabilities and delivery efficiency of LNPs in vivo. Summary of the Invention

[0005] This invention provides a lipid compound, its preparation method and application, and a pharmaceutical composition. The lipid compound of this invention achieves liver-specific expression of mRNA and has high delivery efficiency without relying on any targeting ligand modification.

[0006] This invention provides a lipid compound, which is a compound of Formula I or a stereoisomer, tautomer, solvent compound or pharmaceutically acceptable salt thereof; Formula I; R1 includes a first alkyl or hydroxyl-substituted alkyl group; R2 includes a second alkyl group; R3 includes -H or -OH; G1 includes the first alkylene group; G2 includes a second alkylene group or -O-teralkylene group.

[0007] Preferably, the first alkyl group includes an ethyl group; The hydroxylated alkyl group includes hydroxylated ethyl groups; The second alkyl group has 11 to 17 carbon atoms; The first alkylene group includes pentylene or hexylene; The second alkylene group includes an ethylene group; the third alkylene group includes a pentylene group.

[0008] Preferably, the second alkyl group comprises the structural formula shown in any one of formulas 1 to 3: Formula 1; Formula 2; Formula 3.

[0009] Preferably, the structural formula of the compound represented by Formula I includes one of Formulas I-1 to I-10: Formula I-1; Formula I-2; Formula I-3; Formula I-4; Formula I-5; Formula I-6; Formula I-7; Formula I-8; Formula I-9; Formula I-10.

[0010] The present invention also provides a method for preparing the lipid compound described in the above technical solution, comprising the following steps: Compound A, compound B, a first acid-binding agent, a first catalyst, and a first organic solvent were mixed and subjected to a first nucleophilic substitution reaction to obtain compound C. The structure of compound A is shown in Formula A; the structure of compound B is shown in Formula B; the structure of compound C is shown in Formula C. Formula A; Formula B; Formula C; Compound C, compound D, a second acid-binding agent, a second catalyst, and a second organic solvent were mixed and subjected to a second nucleophilic substitution reaction to obtain the compound shown in Formula I. The structure of compound D is shown in formula D; Formula D; or Compound A, compound E, and an alcohol solvent were mixed and subjected to an epoxy ring-opening reaction to obtain compound F; The structure of compound E is shown in formula E; the structure of compound F is shown in formula F; Formula E; Formula F; Compound F, compound B, a third acid-binding agent, a third catalyst, and an organic solvent were mixed and subjected to an N-alkylation reaction to obtain the compound shown in Formula I.

[0011] Preferably, the molar ratio of compound A to compound B is 1~2:0.8~1; The temperature of the first nucleophilic substitution reaction is 25~100℃, and the time is 12~48 h; The preferred molar ratio of compound C to compound D is 0.13~1:0.46~3.5; The second nucleophilic substitution reaction is carried out at a temperature of 25~100℃ for a time of 12~48 h; The molar ratio of compound A to compound E is 1~2:0.8~1.

[0012] Preferably, the epoxy ring-opening reaction is carried out at a temperature of 25~40℃ for 12~48 h. The molar ratio of compound F to compound B is 0.8~1:1~1.5; The N-alkylation reaction is carried out at a temperature of 25~100℃ for a time of 12~48 h.

[0013] The present invention also provides the application of the compounds described in the above technical solutions or the compounds prepared by the preparation methods described in the above technical solutions in the preparation of liver-targeting drugs.

[0014] The present invention also provides a pharmaceutical composition, characterized in that it comprises the lipid compound described in the above technical solution or the lipid compound prepared by the preparation method described in the above technical solution, neutral lipids, structured lipids and PEG-lipids; The molar ratio of the lipid compound, neutral lipid, structured lipid and PEG-lipid is (0~50):(5~80):(5~50):(0.1~10), and the amount of the lipid compound cannot be 0.

[0015] Preferably, the neutral lipids include one or more of 1,2-distearyl-sn-glycero-3-phosphate choline, 1,2-dispalmitoyl-sn-glycero-3-phosphate choline, 1,2-dispalmitoyl-sn-glycero-3-phosphate ethanolamine, 1,2-dimyristoyl-sn-glycero-3-phosphate choline, 1,2-dimyristoyl-sn-glycero-3-phosphate ethanolamine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate choline, 1,2-dioleoyl-sn-glycero-3-phosphate choline, 2-dioleoyl-sn-glycero-3-phosphate-(1'-rac-glycerol), 1,2-dioleoyl-sn-glycero-3-phosphate ethanolamine, and sphingomyelin; The structural lipids include one or more of cholesterol, coccosterol, sitosterol, ergosterol, campesterol, stigmasterol, and rapeseed sterol; The PEG-lipids include one or more of the following: 2-[(polyethylene glycol)-2000]-N,N-tetracosylacetamide, 1,2-dimyristoyl-sn-glycerol methoxy polyethylene glycol, 1,2-distearate-sn-glycerol-3-phosphate ethanolamine-N-[amino(polyethylene glycol)], PEG-disterol glycerol, PEG-dipalmitoyl, PEG-dioleoyl, PEG-distearate, PEG-diacylglycerol amide, PEG-dipalmitoylphosphatidylethanolamine, PEG-phosphatidylethanolamine, PEG-succinate diacylglycerol, PEG-ceramide, PEG-dialkoxypropylcarbamate, and PEG-1,2-dimyristoyloxypropyl-3-amine; The pharmaceutical composition also includes bioactive ingredients.

[0016] This invention achieves specific expression and efficient delivery of mRNA in the liver by innovatively designing the chemical structure of lipid molecules, especially by specifically modifying the hydrophilic head group and / or hydrophobic tail structure, effectively avoiding off-target effects in non-target organs. It has significant scientific value and broad application prospects. Attached Figure Description

[0017] Figure 1 Bioluminescence results of mice in vivo and in vitro in various organs for LNP-2 / Fluc-mRNA, LNP-5 / Fluc-mRNA, LNP-7 / Fluc-mRNA, LNP-9 / Fluc-mRNA, and LNP-10 / Fluc-mRNA formulations; Figure 2 Bioluminescence results of in vivo and in vitro organs of ai9 mice injected with LNP-7 / Cre-mRNA formulation; Figure 3Immunofluorescence results of frozen sections of liver tissue from ai9 mice injected with LNP-7 / Cre-mRNA and PBS; Figure 4 The results of in vivo bioluminescence in mice injected with LNP-7 / Fluc-mRNA and SM-102 / Fluc-mRNA at 6h, 12h, and 24h. Figure 5 In vivo bioluminescence quantification at 6h, 12h, and 24h in mice injected with LNP-7 / Fluc-mRNA and SM-102 / Fluc-mRNA formulations. Figure 6 The in vitro bioluminescence results of various organs in mice injected with LNP-7 / Fluc-mRNA and SM-102 / Fluc-mRNA at 6h, 12h, and 24h; Figure 7 In vitro bioluminescence quantification of various organs in mice injected with LNP-7 / Fluc-mRNA and SM-102 / Fluc-mRNA at 6h, 12h, and 24h. Figure 8 H&E, Masson, Sirius red staining results for bleomycin-induced pulmonary fibrosis in mice treated with LNP-7 / Anti-IL-11-scfv mRNA formulation; Figure 9 The results of α-SMA immunofluorescence staining of frozen lung sections from a mouse model of bleomycin-induced pulmonary fibrosis treated with LNP-7 / Anti-IL-11-scfv mRNA were obtained. Figure 10 A quantitative graph showing the hydroxyproline content in the lungs of a mouse model of bleomycin-induced pulmonary fibrosis treated with LNP-7 / Anti-IL-11-scfv mRNA. Detailed Implementation

[0018] This invention provides a lipid compound, which is a compound of Formula I or a stereoisomer, tautomer, solvent compound or pharmaceutically acceptable salt thereof; Formula I; R1 includes a first alkyl or hydroxyl-substituted alkyl group; R2 includes a second alkyl group; R3 includes -H or -OH; G1 includes the first alkylene group; G2 includes a second alkylene group or -O-teralkylene group.

[0019] In this invention, the first alkyl group preferably includes an ethyl group; the hydroxylated alkyl group preferably includes a hydroxylated ethyl group; the first alkylene group preferably includes a pentylene or a hexylene; the second alkylene group preferably includes an ethylene; and the third alkylene group preferably includes a pentylene.

[0020] In this invention, the second alkyl group preferably has 11 to 17 carbon atoms, and more preferably includes the structural formula shown in any one of formulas 1 to 3: Formula 1; Formula 2; Formula 3.

[0021] In this invention, the structural formula of Formula I is preferably one of Formulas I-1 to I-10: Formula I-1; Formula I-2; Formula I-3; Formula I-4; Formula I-5; Formula I-6; Formula I-7; Formula I-8; Formula I-9; Formula I-10.

[0022] The present invention also provides a method for preparing the lipid compound described in the above technical solution, comprising the following steps: Compound A, compound B, a first acid-binding agent, a first catalyst, and a first organic solvent were mixed and subjected to a first nucleophilic substitution reaction to obtain compound C. The structure of compound A is shown in Formula A; the structure of compound B is shown in Formula B; the structure of compound C is shown in Formula C. Formula A; Formula B; Formula C; Compound C, compound D, a second acid-binding agent, a second catalyst, and a second organic solvent were mixed and subjected to a second nucleophilic substitution reaction to obtain the compound shown in Formula I. The structure of compound D is shown in formula D; Formula D; or Compound A, compound E, and an alcohol solvent were mixed and subjected to an epoxy ring-opening reaction to obtain compound F; The structure of compound E is shown in formula E; the structure of compound F is shown in formula F; Formula E; Formula F; Compound F, compound B, a third acid-binding agent, a third catalyst, and an organic solvent were mixed and subjected to an N-alkylation reaction to obtain the compound shown in Formula I.

[0023] There are two methods for preparing the lipid compounds of this invention. The first method is described below: In this invention, compound A, compound B, a first acid-binding agent, a first catalyst, and a first organic solvent are mixed to carry out a first nucleophilic substitution reaction to obtain compound C.

[0024] In this invention, the molar ratio of compound A to compound B is preferably 1~2:0.8~1, specifically it can be 1.2:0.8, 1.5:0.8, 1.7:0.8, 2:0.8, 1.2:0.9, 1.5:0.9, 1.7:0.9, 2:0.9, 1.2:1, 1.7:1 or 2:1.

[0025] In this invention, the molar ratio of compound A to the first acid-binding agent is 1:2, and the molar ratio of the first acid-binding agent to the first catalyst is preferably 7.5:1. The first acid-binding agent is preferably potassium carbonate, and the first catalyst preferably includes potassium iodide. The acid-binding agent can neutralize the acidic byproducts generated during the reaction.

[0026] In this invention, the first organic solvent preferably includes MeCN.

[0027] In this invention, the temperature of the first nucleophilic substitution reaction is preferably 25~100℃, specifically 25℃, 30℃, 40℃, 50℃, 60℃, 70℃, 80℃, 90℃ or 100℃, and the time is preferably 12~48 h, specifically 12 h, 24 h, 36 h or 48 h; the first nucleophilic substitution reaction is preferably carried out under stirring conditions.

[0028] After the first nucleophilic substitution reaction, the present invention preferably filters the obtained product, and then removes the solvent from the obtained wet solid before performing column separation.

[0029] After obtaining compound C, the present invention mixes compound C, compound D, a second acid-binding agent, a second catalyst, and a second organic solvent to carry out a second nucleophilic substitution reaction to obtain the compound shown in Formula I.

[0030] In this invention, the molar ratio of compound C to compound D is preferably 0.13~1:0.46~3.5, specifically it can be 0.13:0.46, 0.13:1, 0.13:2, 0.13:3, 0.13:3.5, 0.4:0.46, 0.4:1, 0.4:2, 0.4:3, 0.4:3.5, 0.7:1, 0.7:2, 0.7:3, 0.7:3.5, 1:1, 1:2, 1:3 or 1:3.5.

[0031] In this invention, the molar ratio of compound C to the second acid-binding agent is 1:3, and the molar ratio of the second acid-binding agent to the second catalyst is preferably 7.5:1. The second acid-binding agent is preferably potassium carbonate, and the second catalyst preferably comprises potassium iodide. The acid-binding agent can neutralize the acidic byproducts generated during the reaction.

[0032] In this invention, the second organic solvent preferably includes MeCN.

[0033] In this invention, the temperature of the second nucleophilic substitution reaction is preferably 25~100℃, specifically 25℃, 30℃, 40℃, 50℃, 60℃, 70℃, 80℃, 90℃ or 100℃, and the reaction time is preferably 12~48 h, specifically 12 h, 24 h, 36 h or 48 h; the second nucleophilic substitution reaction is preferably carried out under stirring conditions.

[0034] After the second nucleophilic substitution reaction, the present invention preferably filters the obtained product, and then removes the solvent from the obtained wet solid before performing column separation.

[0035] The second preparation method is described below: Compound A, compound E, and an alcohol solvent were mixed and subjected to an epoxy ring-opening reaction to obtain compound F.

[0036] In this invention, the molar ratio of compound A to compound E is preferably 1~2:0.8~1, specifically it can be 1.2:0.8, 1.5:0.8, 1.7:0.8, 2:0.8, 1.2:0.9, 1.5:0.9, 1.7:0.9, 2:0.9, 1.2:1, 1.7:1 or 2:1.

[0037] In this invention, the alcohol solvent preferably includes ethanol.

[0038] In this invention, the preferred temperature for the epoxy ring-opening reaction is 25~40℃, specifically 25℃, 28℃, 30℃, 33℃, 35℃ or 40℃, and the preferred time is 12~48 h, specifically 12 h, 24 h, 36 h or 48 h; the epoxy ring-opening reaction is preferably carried out under stirring conditions.

[0039] After the epoxy ring-opening reaction, the present invention preferably removes the solvent from the obtained product and then performs column separation.

[0040] After obtaining compound F, the present invention mixes compound F, compound B, a third acid-binding agent, a third catalyst, and an organic solvent to carry out an N-alkylation reaction to obtain the compound shown in Formula I.

[0041] In this invention, the molar ratio of compound F to compound B is preferably 0.8~1:1~1.5, specifically 0.8:1, 0.8:1.1, 0.8:1.2, 0.8:1.3, 0.8:1.4, 0.8:1.5, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, and 1:1.5.

[0042] In this invention, the molar ratio of compound F to the third acid-binding agent is 1:3, and the molar ratio of the third acid-binding agent to the third catalyst is preferably 7.5:1. The third acid-binding agent is preferably potassium carbonate, and the third catalyst preferably includes potassium iodide. The acid-binding agent can neutralize the acidic byproducts generated during the reaction.

[0043] In this invention, the third organic solvent preferably includes MeCN.

[0044] In this invention, the temperature of the N-alkylation reaction is preferably 25~100 °C, specifically 25 °C, 30 °C, 40 °C, 50 °C, 60 °C, 70 °C, 80 °C, 90 °C or 100 °C, and the time is preferably 12~48 h, specifically 12 h, 24 h, 36 h or 48 h; the N-alkylation reaction is preferably carried out under stirring conditions.

[0045] After the N-alkylation reaction, the present invention preferably filters the obtained product, and then removes the solvent from the obtained wet solid before performing column separation.

[0046] The present invention also provides the application of the compounds described in the above technical solutions or the compounds prepared by the preparation methods described in the above technical solutions in the preparation of liver-targeting drugs.

[0047] The present invention also provides a pharmaceutical composition comprising the lipid compound described in the above technical solution or the lipid compound prepared by the preparation method described in the above technical solution, neutral lipids, structured lipids, and PEG-lipids; The molar ratio of the lipid compound, neutral lipid structure lipid, and PEG-lipid is (0~50):(5~80):(5~50):(0.1~10), and the amount of the lipid compound cannot be 0. In a specific embodiment of the present invention, it can be (10~40):(50~70):(5~20):(0.5~5).

[0048] In this invention, the neutral lipids preferably include one or more of the following: 1,2-distearatel-sn-glycero-3-phosphocholine, 1,2-dispalmitoyl-sn-glycero-3-phosphocholine, 1,2-dispalmitoyl-sn-glycero-3-phosphoethanolamine, 1,2-dimyristoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphocholine, 2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, and sphingomyelin. The structural lipids preferably include one or more of cholesterol, coccosterol, sitosterol, ergosterol, campesterol, stigmasterol, and rapeseed sterol; The PEG-lipids preferably include one or more of 2-[(polyethylene glycol)-2000]-N,N-tetracosylacetamide, 1,2-dimyristoyl-sn-glycerol methoxy polyethylene glycol, 1,2-distearate-sn-glycerol-3-phosphate ethanolamine-N-[amino(polyethylene glycol)], PEG-disterol glycerol, PEG-dipalmitoyl, PEG-dioleoyl, PEG-distearate, PEG-diacylglycerol amide, PEG-dipalmitoylphosphatidylethanolamine, PEG-phosphatidylethanolamine, PEG-succinate diacylglycerol, PEG-ceramide, PEG-dialkoxypropylcarbamate, and PEG-1,2-dimyristoyloxypropyl-3-amine.

[0049] In this invention, the pharmaceutical composition preferably further includes a bioactive ingredient; the bioactive ingredient is preferably negatively charged or hydrophobic, and the bioactive ingredient preferably includes one or more of nucleic acid molecules, proteins, peptides and small molecule drugs; the nucleic acid molecule preferably includes messenger RNA.

[0050] The lipid compounds, their preparation methods, applications, and pharmaceutical compositions provided by the present invention are described in detail below with reference to the embodiments. However, these should not be construed as limiting the scope of protection of the present invention.

[0051] Preparation of epoxides Step I: In a round-bottom flask, dissolve raw material (I) (20 mmol) in chloroform (20 mL), add NaOH (20 mmol) at room temperature and stir. Raise the reaction temperature to 45 °C, add raw material (II) (20 mmol) dropwise, and stir for 24 h. When the reaction is complete as monitored by TLC, wash the mixture with water, extract with ethyl acetate, combine the organic layers, dry with anhydrous sodium sulfate, remove the solvent under reduced pressure using a rotary evaporator, and purify by column chromatography (silica gel column 200-300 mesh, eluent PE:EA = 10:1), to obtain transparent liquid III (i.e., raw material (III)). Step II: Dissolve raw material (III) (2.4 mmol) in DCM (10 mL), add DMAP (3.6 mmol), and add phenyl p-nitrochloroformate (2.9 mmol) in portions. Stir at room temperature for 3 h. Add raw material (IV) (2.6 mmol), stir at room temperature for 12 h, dilute with DCM, wash with brine, collect the organic phase, dry with anhydrous sodium sulfate, concentrate and pass through a column (silica gel column 200-300 mesh, eluent PE:EA = 20:1) to obtain yellow transparent liquid V.

[0052] Example 1: Preparation of ionizable lipids Step I: In a round-bottom flask, dissolve starter (I) (1.95 mmol) and starter (II) (1.3 mmol) in 5 mL of anhydrous ethanol, stir at room temperature for 18 h, monitor the reaction until it is complete by TLC, evaporate the solvent to dryness, and separate by column chromatography (silica gel column 200-300 mesh, eluent DCM:MeOH=100:3) to obtain compound III.

[0053] Step II: In a round-bottom flask, compound III (1 mmol), K₂CO₃ (3 mmol), and KI (0.4 mmol) were added sequentially, dissolved in 5 mL of MeCN, and finally compound IV (1.5 mmol) was added. The mixture was stirred at 70°C for 12 h. The reaction was monitored by TLC until completion. The mixture was then filtered, the solvent removed under reduced pressure, and purified by column chromatography (200-300 mesh silica gel column, eluent: DCM:MeOH = 100:3), yielding a transparent, ionizable lipid I-1. The synthesized product was dissolved in CDCl₃ and purified by H₂... 1 -NMR was used to analyze the structure of the product.

[0054] Compound I-1: 1 H NMR (400 MHz, Chloroform- d ) δ 5.05 (t, J= 8.6 Hz, 1H),4.85 – 4.81 (m, 1H), 4.14 – 4.08 (m, 4H), 3.81 – 3.71 (m, 1H), 3.44 (t, J = 6.4Hz, 2H), 3.39 (d, J = 5.0 Hz, 2H), 2.66 – 2.61 (m, 1H), 2.56 – 2.38 (m, 5H), 2.28 – 2.21 (m, 2H), 1.94 (s, 2H), 1.67 – 1.57 (m, 14H), 1.52 – 1.42 (m, 9H),1.41 – 1.10 (m, 17H), 1.01 (t, J = 6.9 Hz, 3H), 0.93 – 0.79 (m, 9H). Examples 2-5 Replacing IV in the above synthetic steps allows the synthesis of compounds I-2 to I-5, with the following 1H NMR spectral data: I-2: 1 H NMR (400 MHz, Chloroform- d ) δ 5.07 (t, J = 7.0 Hz, 1H), 4.88 –4.81 (m, 1H), 4.13 (dt, J = 18.0, 6.4 Hz, 4H), 3.83 – 3.79 (m, 1H), 3.45 (t, J =6.4 Hz, 2H), 3.40 (d, J = 5.0 Hz, 2H), 2.71 – 2.64 (m, 1H), 2.59 – 2.52 (m,2H), 2.49 – 2.45 (m, 2H), 2.27 (t, J = 7.6 Hz, 2H), 2.04 – 1.90 (m, 2H), 1.71 –1.58 (m, 14H), 1.49 – 1.34 (m, 9H), 1.33 – 1.14 (m, 30H), 1.03 (t, J = 7.2 Hz, 3H), 0.91 – 0.83 (m, 9H). I-3: 1 H NMR (400 MHz, Chloroform- d ) δ 5.06 (t,J = 7.0 Hz, 1H), 4.88 –4.81 (m, 1H), 4.12 (dt, J = 18.0, 6.4 Hz, 4H), 3.90 – 3.86 (m, 1H), 3.45 (t, J =6.4 Hz, 2H), 3.41 (d, J = 5.2 Hz, 2H), 2.77 – 2.63 (m, 1H), 2.66 – 2.55 (m,4H), 2.26 (t, J = 7.6 Hz, 2H), 2.04 – 1.87 (m, 1H), 1.69 – 1.41 (m, 24H), 1.32– 1.24 (m, 20H), 1.03 (t, J = 7.2 Hz, 3H), 0.90 – 0.86 (m, 9H). I-4: 1 H NMR (400 MHz, Chloroform- d ) δ 5.05 (t, J = 7.0 Hz, 1H), 4.89 –4.79 (m, 1H), 4.12 (dt, J = 18.0, 6.4 Hz, 4H), 3.88 – 3.86 (m, 1H), 3.44 (t, J =6.4 Hz, 2H), 3.41 (d, J = 5.6 Hz, 2H), 2.78 – 2.68 (m, 1H), 2.66 – 2.45 (m,5H), 2.24 (t, J = 7.4 Hz, 2H), 2.04 – 1.87 (m, 1H), 1.70 – 1.36 (m, 23H), 1.35– 1.15 (m, 24H), 1.07 (t, J = 7.2 Hz, 3H), 0.92 – 0.80 (m, 9H). I-5: 1 H NMR (400 MHz, Chloroform- d ) δ 5.06 (t, J= 6.8 Hz, 1H), 4.87 –4.81 (m, 1H), 4.20 – 4.06 (m, 4H), 3.84 – 3.81 (m, 1H), 3.45 (t, J = 6.4 Hz, 2H), 3.40 (d, J = 5.0 Hz, 2H), 2.71 – 2.60 (m, 1H), 2.59 – 2.46 (m, 5H), 2.25(t, J = 7.4 Hz, 2H), 2.03 – 1.89 (m, 1H), 1.72 – 1.39 (m, 24H), 1.33 – 1.18 (m,31H), 1.04 (t, J = 7.2 Hz, 3H), 0.91 – 0.82 (m, 9H). Preparation of brominated carbonates In a round-bottom flask, compound I (30 mmol) was dissolved in 30 mL of DCM and cooled to 0 °C. Then, DMAP (45 mmol) and phenyl p-nitrochloroformate (33 mmol) were added, and the mixture was stirred at room temperature for 4 h. Finally, compound I was added, and the mixture was allowed to react overnight at room temperature. The reaction was monitored by TLC until completion. The solvent was removed under reduced pressure, and the mixture was purified by column chromatography (200-300 mesh silica gel column, eluent: PE:EA = 20:1) to obtain a yellow liquid compound III.

[0055] Preparation of bromoalkyl esters In a round-bottom flask, compound I (11 mmol), compound II (10 mmol), EDCI (15 mmol), and DMAP (2 mmol) were added sequentially and dissolved in 30 mL of DCM. The mixture was stirred at room temperature for 4 h, and the reaction was monitored by TLC until completion. The solvent was removed under reduced pressure, and the mixture was purified by column chromatography (200-300 mesh silica gel column, eluent: PE:EA = 20:1) to obtain a transparent liquid compound III.

[0056] Example 6 Step I: In a round-bottom flask, compound I (7.5 mmol), K₂CO₃ (15 mmol), and KI (2 mmol) were added sequentially, dissolved in 20 mL MeCN, and finally compound II (5 mmol) was added. The mixture was stirred at 70 °C for 12 h. The reaction was monitored by TLC until it was complete. The solvent was removed by filtration and reduced pressure, and the mixture was purified by column chromatography (200-300 mesh silica gel column, eluent: DCM:MeOH = 20:1), yielding a light yellow transparent liquid intermediate III.

[0057] Step II: In a round-bottom flask, compound III (1 mmol), K₂CO₃ (3 mmol), and KI (0.4 mmol) were added sequentially, dissolved in 5 mL of MeCN, and finally compound IV (1.5 mmol) was added. The mixture was stirred at 70 °C for 12 h. The reaction was monitored by TLC until it was complete. The mixture was then filtered, the solvent was removed under reduced pressure, and the solution was purified by column chromatography (200-300 mesh silica gel column, eluent: DCM:MeOH = 100:3), yielding a yellow, transparent, ionizable lipid I-6.

[0058] The synthesized product was dissolved in CDCl3 and subjected to H2... 1 -NMR was used to analyze the structure of the product.

[0059] I-6: 1 H NMR (400 MHz, Chloroform- d ) δ 5.08 (t, J = 8.6 Hz, 1H), 4.86 (p, J = 6.1 Hz, 1H), 4.22 – 4.09 (m, 4H), 3.77 (t, J = 4.4 Hz, 2H), 2.87 (s, 2H), 2.77 (t, J = 7.4 Hz, 4H), 2.30 (t, J = 7.3 Hz, 2H), 2.07 – 1.87 (m, 2H), 1.74 –1.63 (m, 11H), 1.60 (s, 3H), 1.55 – 1.46 (m, 5H), 1.42 – 1.16 (m, 20H), 0.94– 0.84 (m, 9H). Example 7 Replacing IV in Example 6 allows for the synthesis of I-7 to I-10, with the following proton NMR spectral data: I-7: 1 H NMR (400 MHz, Chloroform- d) δ 5.08 (t, J = 7.3 Hz, 1H), 4.86 (p, J = 6.3 Hz, 1H), 4.22 – 4.08 (m, 4H), 3.74 (t, J = 4.4 Hz, 2H), 2.83 (s, 2H),2.72 (s, 4H), 2.30 (t, J = 7.5 Hz, 2H), 2.03 – 1.91 (m, 2H), 1.77 – 1.54 (m,16H), 1.54 – 1.46 (m, 5H), 1.37 – 1.18 (m, 30H), 0.93 – 0.85 (m, 9H). I-8: 1 H NMR (400 MHz, Chloroform- d ) δ 5.08 (t, J = 8.4 Hz, 1H), 4.86 (p, J = 6.3 Hz, 1H), 4.19 – 4.10 (m, 4H), 3.81 (t, J = 4.4 Hz, 2H), 2.91 (s, 2H),2.81 (s, 4H), 2.29 (t, J = 7.4 Hz, 2H), 2.03 – 1.92 (m, 2H), 1.76 – 1.58 (m,16H), 1.52 – 1.42 (m, 6H), 1.37 – 1.23 (m, 20H), 0.93 – 0.85 (m, 9H). I-9: 1 H NMR (400 MHz, Chloroform- d ) δ 5.08 (t, J = 7.1 Hz, 1H), 4.86 (p, J = 6.2 Hz, 1H), 4.21 – 4.10 (m, 4H), 3.77 (t, J = 4.5 Hz, 2H), 2.86 (s, 2H),2.76 (s, 4H), 2.28 (t, J= 7.4 Hz, 2H), 2.02 – 1.89 (m, 2H), 1.76 – 1.57 (m,16H), 1.51 – 1.40 (m, 6H), 1.37 – 1.22 (m, 24H), 0.92 – 0.85 (m, 9H). I-10: 1 H NMR (400 MHz, Chloroform- d ) δ 5.08 (t, J = 7.1 Hz, 1H), 4.85 (p, J = 6.2 Hz, 1H), 4.20 – 4.09 (m, 4H), 3.80 (t, J = 4.5 Hz, 2H), 2.90 (s, 2H), 2.79 (s, 4H), 2.28 (t, J = 7.4 Hz, 2H), 2.02 – 1.91 (m, 2H), 1.75 – 1.57 (m,16H), 1.53 – 1.43 (m, 6H), 1.40 – 1.20 (m, 33H), 0.93 – 0.85 (m, 9H). Example 8 Firefly luciferase (Flu, purchased from Jiangsu Genscript Biotech Co., Ltd.) was selected as the reporter gene marker mRNA, namely Flu mRNA. LNPs / mFluc (LNPs loaded with Flu mRNA) were prepared, and their formulation properties such as particle size, potential, and encapsulation efficiency were investigated to evaluate their formulation properties.

[0060] The preparation method of LNPs / mFluc is as follows: (1) Solution preparation: 300 µg of the prepared ionizable lipids (I-1~I-10) were dissolved together with DSPC, cholesterol and PEGylated lipids (DMG-PEG2000) in 0.5 mL of anhydrous ethanol to form a homogeneous lipid ethanol solution, which served as the ethanol phase. At the same time, 20 µg of Fluc mRNA was dissolved in 1.5 mL of 50 mM (pH=4) citrate buffer solution, which served as the aqueous phase. The mass ratio of ionizable lipids to mRNA was 50:1, and the mass ratio of each component in the formulation was: ionizable lipids:DSPC:cholesterol:DMG-PEG2000 = 50:38.5:10:1.5.

[0061] (2) LNP preparation: The ethanol phase and aqueous phase obtained in step (1) were mixed at a volume ratio of 1:3 to obtain the initial LNPs / mFluc formulation. The flow rate of the ethanol phase was 3 mL / min and the flow rate of the aqueous phase was 9 mL / min.

[0062] (3) Ultrafiltration: The initial LNPs / mFluc formulation obtained in step (2) was placed in a dialysis bag and dialyzed in PBS buffer solution (prepared with RNase-free water) at pH 7.4 for 8 hours to collect the ethanol-free LNPs / mFluc formulation. The dialyzed formulation was concentrated by ultrafiltration through an ultrafiltration tube to obtain the LNPs / mFluc formulation (denoted as LNP-1~LNP-11; taking LNP-1 as an example, LNP-1 refers to the lipid nanoparticles prepared by I-1).

[0063] LNPs / mFluc formulation property study: Particle size, potential, and PDI determination: The particle size and potential of LNPs / mFluc were determined using a Malvern laser particle size analyzer. A certain volume of freshly prepared LNPs / mFluc formulation was diluted 10-fold with PBS buffer at pH 7.4, and its particle size, potential, and PDI were determined using a Malvern laser particle size analyzer. Each sample was measured in triplicate at a temperature of 25℃. The results are shown in Table 1.

[0064] Encapsulation efficiency testing: using Quant iT TM RiboGreen TM The kit was used to test the encapsulation efficiency of each LNPs / mFluc formulation. The results of representative LNP detection are shown in Table 1.

[0065] Table 1. Parameters of nanoparticles formed by encapsulating Fluc mRNA with lipid nanoparticles

[0066] As shown in Table 1, the average particle size of lipid nanoparticles LNP-2, LNP-5, LNP-7, LNP-9, and LNP-10 is in the range of 80–120 nm, with a PDI of around 0.1. They exhibit uniform particle size distribution and all have a negative charge potential. The encapsulation efficiency of the lipid nanoparticles is above 80%. These five LNPs will be selected for in vivo validation via mfluc delivery.

[0067] The ability of LNPs / mFluc formulations (LNP-2, LNP-5, LNP-7, LNP-9, LNP-10) prepared by the compounds of this invention to express mRNA in vivo was investigated by tail vein administration.

[0068] Two mL of LNP formulation containing 10 μg Fluc-mRNA was injected via the tail vein into four 6-8 week old female C57 mice weighing 18-20 g. Six hours after administration, 100 μL of substrate solution (30 mg / mL, fluorescein potassium salt) was injected intraperitoneally. The mean radiation intensity (corresponding to fluorescence expression intensity) of the protein expressed by the LNP-carried mRNA in mice was then detected using an IVIS small animal in vivo imaging system. The results of the mouse in vivo imaging are shown in the figure below. Figure 1 Mice were euthanized, and their organs—heart, liver, spleen, lung, and kidney—were precisely isolated. The bioluminescence intensity of each isolated organ was measured using an IVIS instrument with an exposure time of 10 s. The results are shown below. Figure 1 .

[0069] Figure 1 Bioluminescence results of mouse in vivo and in vitro organs for LNP-2 / Fluc-mRNA, LNP-5 / Fluc-mRNA, LNP-7 / Fluc-mRNA, LNP-9 / Fluc-mRNA, and LNP-10 / Fluc-mRNA formulations.

[0070] In vivo expression studies via tail vein injection showed that, based on the total bioluminescence amount in the tail vein, the ionizable lipids of this invention can promote higher mRNA expression in mice. Furthermore, LNP-2 / mFluc, LNP-5 / mFluc, LNP-7 / mFluc, LNP-9 / mFluc, and LNP-10 / mFluc, after tail vein injection, can deliver mRNA to the liver, resulting in weak or almost no expression in other organs, achieving liver-specific targeting. LNP-7 showed the best liver-targeting effect.

[0071] Example 9 The only difference from Example 8 is that the firefly luciferase-labeled mRNA was replaced with mRNA encoding Cre recombinase (purchased from Jiangsu Genscript Biotech Co., Ltd.).

[0072] Following the method in Example 8, the mRNA encoding Cre recombinase (mCre) was encapsulated in LNP-7 to form the LNP-7 / mCre complex.

[0073] Ai9 reporter mice (which express tdTomato red fluorescent protein only in the presence of Cre recombinase) were administered 2 mL of LNP-7 / mCre complex formulation containing 20 µg Cre mRNA or PBS solution via tail vein injection. Forty-eight hours after administration, frozen sections of mouse liver tissue were prepared and immunofluorescence stained with the nuclear dye Hoechst 33342.

[0074] Figure 2 Bioluminescence results of in vivo and in vitro organs of ai9 mice injected with LNP-7 / Cre-mRNA formulation; Figure 3 Immunofluorescence results of frozen sections of liver tissue from ai9 mice injected with LNP-7 / Cre-mRNA and PBS.

[0075] Depend on Figures 2-3 It was observed that in the PBS control group, only blue cell nuclei were visible in liver sections stained with Hoechst 33342, with no obvious tdTomato red fluorescence signal, indicating no background expression. In the LNP-7 / mCre experimental group, extensive and strong spontaneous tdTomato red fluorescence signal was observed around the Hoechst 33342-stained cell nuclei, which was highly co-localized with the liver parenchyma. These results clearly demonstrate that the nanoparticles prepared from the ionizable lipid LNP-7 of this invention can efficiently and specifically deliver functional mCre to the mouse liver and successfully translate into bioactive Cre recombinase within hepatocytes, thereby activating reporter gene expression. This fully confirms the application potential of the LNP system in liver-targeted mRNA therapy.

[0076] Example 10 To highlight the superiority of LNP-7 of the present invention over commonly used lipids in the prior art (such as SM-102), this embodiment presents a systematic in vivo bioluminescence imaging comparison.

[0077] The control group differed from Example 8 only in that the lipids were replaced with the SM-102 / mFluc formulation (the preparation method of the SM-102 / mFluc formulation is the same as that of the LNPs / mFluc formulation); and LNP-7 / mFluc, which encapsulates firefly luciferase mRNA (mFluc), was used as the experimental group.

[0078] Healthy mice were given the same doses of the control and experimental formulations via tail vein injection.

[0079] Figure 4 The results of in vivo bioluminescence at 6h, 12h, and 24h in mice injected with LNP-7 / Fluc-mRNA and SM-102 / Fluc-mRNA were presented.

[0080] Figure 5 In vivo bioluminescence quantification at 6h, 12h, and 24h in mice injected with LNP-7 / Fluc-mRNA and SM-102 / Fluc-mRNA formulations.

[0081] Figure 6The in vitro bioluminescence results of various organs in mice injected with LNP-7 / Fluc-mRNA and SM-102 / Fluc-mRNA at 6h, 12h, and 24h were compared.

[0082] Figure 7 In vitro bioluminescence quantification of organs in mice injected with LNP-7 / Fluc-mRNA and SM-102 / Fluc-mRNA at 6h, 12h, and 24h.

[0083] In vivo imaging kinetics: In vivo imaging was performed at 6, 12, and 24 hours post-drug administration. At all time points, the overall bioluminescent signal intensity in the LNP-7 / mFluc group was higher than that in the SM-102 / mFluc group. The signal of LNP-7 / mFluc peaked at 12 hours and remained at a high level at 24 hours, indicating that its mediated protein expression was more potent and longer-lasting.

[0084] Ex vivo tissue distribution: Major organs were harvested 24 hours later for ex vivo imaging. For example... Figure 6 As shown, LNP-7 / fluc still exhibited high protein expression levels 24 hours after transfection, and its mRNA delivery efficiency was significantly better than that of the control lipid SM-102. Furthermore, the signal from the LNP-7 / mFluc group was highly specifically enriched in the liver (>90%), while the signal from other organs was weak (see [link to data]). Figure 7 This comparative experiment demonstrates that the LNP-7 of this invention is superior to the existing lipid SM-102 in terms of mRNA delivery efficiency, expression persistence, and liver targeting specificity.

[0085] Example 11 The delivery system constructed using LNP-7 of this invention can not only efficiently target the liver, but also deliver secretory antibody fragment mRNA, which can express and secrete therapeutic proteins in the liver into the systemic circulation, thereby achieving effective treatment for diseases of distant organs (such as pulmonary fibrosis).

[0086] A pulmonary fibrosis model was established using C57BL / 6 mice via intratracheal infusion of bleomycin. The model mice were randomly divided into three groups: Healthy control group (G1): healthy mice were injected with PBS via the tail vein; Model control group (G2: PBS): mice with an established pulmonary fibrosis model were injected with PBS via the tail vein. Treatment group (G3: LNP-7 / mAnti-IL-11-scfv): Mice with established models were injected via tail vein injection of an LNP-7 formulation (LNP-7 / mAnti IL-11-scfv vaccine) encapsulated with mRNA encoding an anti-IL-11 single-chain antibody (scFv). The plasmid DNA template for the mRNA was designed using SnapGene software and synthesized by GeneScript. The plasmid-encoded fusion protein (Anti IL-11-scfv) construct also included a T7 promoter, a 5′ untranslated region (UTR), a 3′ UTR, and a poly-A tail. The mRNA was co-transcribed with 4 mM CAP GAG (CAP3111, SYNTHGENE) and chemically modified by replacing uracil with N1-Me-pUTP (NMPUTP001, SYNTHGENE). The mRNA was purified by lithium chloride precipitation and cleaned using the Monarch® RNA Cleanup Kit (T2050S, NEB). The mRNA was confirmed by agarose gel electrophoresis. To ensure the integrity of the sample, it was stored at -80°C.

[0087] Intravenous administration was initiated at a designated time point after model establishment, twice weekly for a total of 3 weeks. After treatment, lung tissue was harvested for H&E, Masson's trichrome, and Picosirius Red staining to assess the degree of fibrosis.

[0088] Figure 8 H&E, Masson, Sirius red staining results for a mouse bleomycin-induced pulmonary fibrosis treatment model induced by injection of LNP-7 / Anti-IL-11-scfv mRNA.

[0089] Figure 9 The results of α-SMA immunofluorescence staining of frozen lung sections from a mouse model of bleomycin-induced pulmonary fibrosis treated with LNP-7 / Anti-IL-11-scfv mRNA.

[0090] Figure 10 A quantitative graph showing the hydroxyproline content in the lungs of a mouse model of bleomycin-induced pulmonary fibrosis treated with LNP-7 / Anti-IL-11-scfv mRNA.

[0091] In the G2 (PBS) group, mice exhibited extensive inflammatory cell infiltration, severe alveolar structural damage, and widespread blue (Masson's red) or red (Sirius red) collagen fiber deposition in their lung tissue, displaying typical fibrotic pathological features. In the G3 (LNP-7 / mAnti-IL-11-scfv) treatment group, the pathological changes in lung tissue were significantly reduced, alveolar structure remained relatively intact, inflammatory infiltration decreased, and both the area and density of collagen fiber deposition were significantly reduced. Figure 8 This indicates that the anti-IL-11 scFv expression mediated by the delivery system of this invention can effectively inhibit the progression of pulmonary fibrosis. The extent and intensity of α-SMA positive signals in the lung tissue of the G3 treatment group were significantly reduced. Figure 9 This indicates that anti-IL-11 scFv treatment effectively inhibits the activation and proliferation of myofibroblasts. The total collagen content in lung tissue was quantitatively analyzed using a hydroxyproline content assay. The hydroxyproline content in the lung tissue of mice in the G3 (LNP-7 / mAnti-IL-11-scfv) treatment group was statistically significantly lower than that in the G2 (PBS) control group. Figure 10 The quantitative data are consistent with the tissue staining results, directly confirming at the biochemical level that the treatment strategy of this invention can effectively reduce excessive collagen deposition in the pulmonary fibrosis model.

[0092] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A lipid compound, characterized in that, The compound represented by Formula I or its stereoisomers, tautomers, solvent compounds or pharmaceutically acceptable salts thereof; Equation I; R1 includes a first alkyl or hydroxyl-substituted alkyl group; R2 includes a second alkyl group; R3 includes -H or -OH; G1 includes the first alkylene group; G2 includes a second alkylene group or -O-teralkylene group.

2. The lipid compound according to claim 1, characterized in that, The first alkyl group includes ethyl; The hydroxylated alkyl group includes hydroxylated ethyl groups; The second alkyl group has 11 to 17 carbon atoms; The first alkylene group includes pentylene or hexylene; The second alkylene group includes an ethylene group; the third alkylene group includes a pentylene group.

3. The lipid compound according to claim 1 or 2, characterized in that, The second alkyl group includes the structural formula shown in any one of formulas 1 to 3: Formula 1; Formula 2; Formula 3.

4. The lipid compound according to claim 1, characterized in that, The structural formula of the compound represented by Formula I includes one of Formulas I-1 to I-10: Equation I-1; Equation I-2; Equation I-3; Equation I-4; Equation I-5; Formula I-6; Equation I-7; Equation I-8; Formula I-9; Formula I-10.

5. A method for preparing the lipid compound according to any one of claims 1 to 4, characterized in that, Includes the following steps: Compound A, compound B, a first acid-binding agent, a first catalyst, and a first organic solvent were mixed and subjected to a first nucleophilic substitution reaction to obtain compound C. The structure of compound A is shown in Formula A; the structure of compound B is shown in Formula B; the structure of compound C is shown in Formula C. Formula A; Formula B; Formula C; Compound C, compound D, a second acid-binding agent, a second catalyst, and a second organic solvent were mixed and subjected to a second nucleophilic substitution reaction to obtain the compound shown in Formula I. The structure of compound D is shown in formula D; Formula D; or Compound A, compound E, and an alcohol solvent were mixed and subjected to an epoxy ring-opening reaction to obtain compound F; The structure of compound E is shown in formula E; the structure of compound F is shown in formula F; Formula E; Formula F; Compound F, compound B, a third acid-binding agent, a third catalyst, and an organic solvent were mixed and subjected to an N-alkylation reaction to obtain the compound shown in Formula I.

6. The preparation method according to claim 5, characterized in that, The molar ratio of compound A to compound B is 1~2:0.8~1; The temperature of the first nucleophilic substitution reaction is 25~100℃, and the time is 12~48 h; The preferred molar ratio of compound C to compound D is 0.13~1:0.46~3.5; The second nucleophilic substitution reaction is carried out at a temperature of 25~100℃ for a time of 12~48 h; The molar ratio of compound A to compound E is 1~2:0.8~1.

7. The preparation method according to claim 5, characterized in that, The epoxy ring-opening reaction is carried out at a temperature of 25~40℃ for a time of 12~48 h. The molar ratio of compound F to compound B is 0.8~1:1~1.5; The N-alkylation reaction is carried out at a temperature of 25~100℃ for a time of 12~48 h.

8. The use of the compound according to any one of claims 1 to 4 or the compound prepared by the preparation method according to any one of claims 5 to 7 in the preparation of a liver-targeting drug.

9. A pharmaceutical composition, characterized in that, Includes lipid compounds as described in any one of claims 1 to 4 or lipid compounds prepared by the preparation method described in any one of claims 5 to 7, neutral lipids, structured lipids, and PEG-lipids; The molar ratio of the lipid compound, neutral lipid, structured lipid and PEG-lipid is (0~50):(5~80):(5~50):(0.1~10), and the amount of the lipid compound cannot be 0.

10. The pharmaceutical composition according to claim 9, characterized in that, The neutral lipids include one or more of the following: 1,2-distearatel-sn-glycero-3-phosphocholine, 1,2-dispalmitoyl-sn-glycero-3-phosphocholine, 1,2-dispalmitoyl-sn-glycero-3-phosphoethanolamine, 1,2-dimyristoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphocholine, 2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, and sphingomyelin. The structural lipids include one or more of cholesterol, coccosterol, sitosterol, ergosterol, campesterol, stigmasterol, and rapeseed sterol; The PEG-lipids include one or more of the following: 2-[(polyethylene glycol)-2000]-N,N-tetracosylacetamide, 1,2-dimyristoyl-sn-glycerol methoxy polyethylene glycol, 1,2-distearate-sn-glycerol-3-phosphate ethanolamine-N-[amino(polyethylene glycol)], PEG-disterol glycerol, PEG-dipalmitoyl, PEG-dioleoyl, PEG-distearate, PEG-diacylglycerol amide, PEG-dipalmitoylphosphatidylethanolamine, PEG-phosphatidylethanolamine, PEG-succinate diacylglycerol, PEG-ceramide, PEG-dialkoxypropylcarbamate, and PEG-1,2-dimyristoyloxypropyl-3-amine; The pharmaceutical composition also includes bioactive ingredients.

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