Guanidyl lipid compound and application thereof

Abstract

The invention discloses a guanidyl lipid compound and application thereof, and belongs to the technical field of medicines. The structural general formula of the compound is as shown in any one of formula (I) to formula (IV), and guanidyl of the molecule is helpful to be combined with mRNA and other nucleic acid drugs and assists nucleic acid molecules in entering cells to play a role. The invention discloses various applications of the guanidyl lipid molecule. On one hand, the molecules can be used as a transfection enhancer to be added into four-component lipid nanoparticles in a small amount, so that the in-vivo and in-vitro mRNA transfection effects of the original lipid nanoparticles are enhanced; on the other hand, the reagent can be used as a single-component transfection reagent to directly deliver mRNA into cells to realize mRNA transfection. Under the applications, the guanidyl lipid molecules show relatively good nucleic acid delivery and transfection capacities, and have important significance in promoting the development of nucleic acid delivery.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a guanidine lipid compound and its applications. Background Technology

[0002] Lipid nanoparticles (LNPs) are a delivery system for small molecules and nucleic acid drugs. In recent years, LNP vaccines delivering mRNA nucleic acid molecules have been extensively studied. Naked RNA is a negatively charged hydrophilic macromolecule that is difficult to enter cells due to electrostatic repulsion of the cell membrane and is easily degraded by RNases in vivo. Encapsulating RNA with lipid vesicles allows it to pass through the cell membrane and release into the cytoplasm.

[0003] Lipid nanoparticles are generally composed of cationic lipids, phospholipids, cholesterol, and polyethylene glycol lipid molecules. The preparation of lipid nanoparticles depends on their self-assembly capability, meaning that lipid components spontaneously organize into nanostructures through intermolecular interactions. First, negatively charged nucleic acids and positively charged lipids bind electrostatically. Then, they assemble through hydrophobic and van der Waals interactions between lipid components, forming lipid nanoparticles (LNPs). Optimizing the structure of lipid nanoparticles can facilitate LNP entry into cells and release mRNA, improving the efficiency of LNP action and playing a significant role in enhancing the preventive effect of vaccines.

[0004] The overall structure of cationic and ionizable lipids can be divided into three parts: (1) head, (2) linker fragment, and (3) tail. The head group of ionizable lipids usually carries a positive charge. The size and charge density of the head group are mainly involved in processes such as encapsulating nucleic acids, stabilizing LNPs, interacting with the cell membrane, and promoting endosome escape. The linker fragment connects the head and tail and can be divided into non-biodegradable (such as ethers and carbamates) and biodegradable (such as esters, amides, and thiols). The hydrophobic tail affects pKa, lipophilicity, fluidity, and fusion properties, thereby affecting the formation and efficacy of LNPs.

[0005] The guanidinyl group is a cationic group and, as a metabolite of guanine, exhibits good biocompatibility. The guanidinyl group can form bidentate hydrogen bonds with phosphate groups in the cell membrane, giving it good ability to penetrate the cell membrane. ChemMedChem (2008, 3, 1635-1643). Current research reports a polymer containing a guanidine side chain with strong membrane-penetrating ability, capable of efficiently delivering fluorescent probes and paclitaxel molecules into cells. J. Am. Chem. Soc. (2016, 138, 3510-3517). Therefore, introducing guanidine groups into lipid nanoparticles has good application potential for improving drug delivery efficiency. Summary of the Invention

[0006] The purpose of this invention is to provide a lipid molecule that is easy to synthesize and can be used to prepare an efficient and safe nucleic acid delivery system.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: A first aspect of the present invention provides a guanidine-based lipid compound, the structural formula of which is shown in any one of formulas (I)-(IV). (I) (II) (III) (IV); R1 and R2 are alkyl chains with 4-17 carbon atoms, respectively.

[0008] The guanidine group in the aforementioned lipid compounds binds to both nucleic acid molecules such as mRNA and phospholipids on the cell membrane, which helps nucleic acid molecules enter the cell and exert their functions.

[0009] Preferably, R1 and R2 are alkyl chains with 7-17 carbon atoms.

[0010] Furthermore, the structural formula of the compound is shown in any one of formulas (A1) to (A5), (B1) to (B3), (C1) to (C4), and (D1) to (D3). (A1); (A2); (A3); (A4); (A5); (B1); (B2); (B3); (C1); (C2); (C3); (C4); (D1); (D2); (D3)

[0011] A second aspect of the present invention provides lipid nanoparticles containing the above-mentioned guanidine lipid compound, wherein the lipid nanoparticles are composed of the guanidine lipid compound, cationic lipids, phospholipids, cholesterol and lipids containing polyethylene glycol.

[0012] In this invention, a guanidine lipid compound is added as a transfection enhancer to lipid nanoparticles to improve their ability to deliver nucleic acid molecules. Specifically, the lipid nanoparticles consist of the guanidine lipid compound, ionizable lipids, phospholipids, cholesterol, and polyethylene glycol-containing lipid molecules. The guanidine lipid compound, ionizable lipids, phospholipids, cholesterol, and polyethylene glycol-containing lipid molecules can self-assemble to form lipid nanoparticles, which can then serve as carriers for encapsulating nucleic acid drugs.

[0013] This invention demonstrates that adding the guanidine lipid compound as a nucleic acid delivery enhancer to lipid nanoparticles results in higher nucleic acid transfection efficiency in both cells and mice compared to commercially available four-component lipid nanoparticles.

[0014] Furthermore, the ionizable lipids can be, but are not limited to, 1-octylnonyl 8-[(2-hydroxyethyl)[6-O-6-(undecyloxy)hexyl]amino]octanoate (SM102), 4-(N,N-dimethylamino)butyrate (dilinyl)methyl ester (MC3), and ((4-hydroxybutyl)azadialkyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315).

[0015] Furthermore, the phospholipid may be, but is not limited to, distearyl phosphatidylcholine (DSPC), 1,2-dioleoyl lecithin (DOPC), myristoyl phosphatidylcholine (DMPC), distearyl phosphatidylethanolamine (DSPE), myristoyl phosphatidylethanolamine (DOPE), and myristoyl phosphatidylethanolamine (DMPE).

[0016] Furthermore, the polyethylene glycol-containing lipid is dimyristylglycerol-polyethylene glycol 2000 (DMG-PEG). 2k ) or methoxy polyethylene glycol bis(tetradecyl acetamide) (ALC-0519).

[0017] Furthermore, the molar amount of the guanidine-based lipid compound accounts for 5-10% of the total molar amount of ionizable lipids, phospholipids, cholesterol, and polyethylene glycol-containing lipids; the molar ratio of ionizable lipids, phospholipids, cholesterol, and polyethylene glycol-containing lipids is 45-50:8-10:35-45:0.5-2. Studies have shown that lipid nanoparticles prepared by adding 5-10% of the guanidine-based lipid compound to commercial four-component lipid nanoparticle formulations significantly improve cell transfection efficiency.

[0018] Preferably, the molar ratio of guanidine lipid compound, ionizable lipid, phospholipid, cholesterol, and polyethylene glycol-containing lipid molecule is 5:50:10:38.5:1.5 or 5:46.3:9.4:42.7:1.6. Under this ratio, the self-assembled lipid nanoparticles have a uniform particle size distribution, good stability, high drug encapsulation efficiency, and high nucleic acid transfection efficiency.

[0019] The lipid nanoparticles can be prepared using, but are not limited to, the following methods: ethanol injection, thin film method, ultrasonic method, and microfluidic method. Specifically, when preparing drug-loaded lipid nanoparticles, the above methods are used to allow the lipid material and negatively charged drugs such as nucleic acids to self-assemble into nanoparticles through supramolecular forces and electrostatic interactions.

[0020] The ethanol injection method involves dissolving guanidine lipid compounds, ionizable lipids, phospholipids, cholesterol, and polyethylene glycol-containing lipid molecules in an appropriate amount of ethanol. The ethanol solution containing lipid materials is then injected into a buffer solution containing drugs, where they self-assemble to form nanoparticles. The ethanol is then removed by dialysis, and the aggregated and precipitated lipid carriers are removed by filtration to obtain stable nanoparticles.

[0021] Compared to commercially available four-component lipid nanoparticles, the addition of the guanidine lipid molecules provided in this invention can enhance the ability of commercially available lipid nanoparticles to deliver nucleic acid molecules such as mRNA into cells.

[0022] A third aspect of the present invention provides the application of the lipid nanoparticles as carriers in the preparation and delivery of nucleic acid drugs.

[0023] Furthermore, the application includes: adding each component to an acidic buffer containing nucleic acid, self-assembling to form lipid nanoparticles carrying nucleic acid, and preparing the nucleic acid delivery drug.

[0024] Furthermore, the nucleic acid is mRNA.

[0025] Furthermore, the total mass ratio of lipid nanoparticles to mRNA was 40:1.

[0026] A fourth aspect of this invention provides the use of the aforementioned guanidine lipid compound as a carrier in the preparation of nucleic acid delivery drugs. The guanidine lipid compound is used as a single-component nucleic acid delivery reagent in the preparation of nucleic acid delivery drugs.

[0027] Furthermore, the application includes: adding the guanidine lipid compound to an acidic buffer containing nucleic acid to form a lipid / nucleic acid complex, thereby preparing the nucleic acid delivery drug.

[0028] Preferably, the molar ratio of guanidine lipid compound to nucleic acid is 5-15:1.

[0029] The beneficial effects of this invention are: (1) This invention provides a series of guanidine lipid molecules. The guanidine group of these molecules can bind to both nucleic acid drugs such as mRNA and phospholipids on the cell membrane, thereby assisting nucleic acid molecules to enter the cell and exert their effects.

[0030] (2) Adding a small amount of the guanidine lipid molecules provided by the present invention to commercial lipid nanoparticles composed of ionizable lipid molecules, phospholipids, cholesterol and lipid molecules containing polyethylene glycol can effectively improve the ability of lipid nanoparticles to deliver mRNA. Therefore, guanidine lipid molecules can be used as an enhancer to improve the delivery efficiency of lipid nanoparticles.

[0031] (3) The guanidine lipid molecule provided by the present invention can be used as a single-component transfection reagent, which can be directly mixed with mRNA to form a complex to achieve intracellular transfection of mRNA, and the effect is better than that of commercial cationic lipid transfection reagents. Attached Figure Description

[0032] Figure 1 The in vitro transfection effect of adding guanidine lipid molecules to lipid nanoparticles SM-LNP is shown in the figure.

[0033] Figure 2 for Figure 1 Quantitative results of fluorescence intensity in the medium.

[0034] Figure 3 Cell transfection effects after adding guanidine lipid molecules to two types of lipid nanoparticles (ALC-LNP and MC3-LNP).

[0035] Figure 4 The cell transfection effect of adding guanidine lipid molecules to SM-LNP in different cell models (BHK, 4T1 and 3T3) was investigated.

[0036] Figure 5 The image shows the mRNA transfection effect in mice after the addition of guanidine lipid molecules to SM-LNP. (A) shows in vivo images of small animals; (B) shows quantitative fluorescence data from in vivo mouse imaging.

[0037] Figure 6 The in vitro transfection effect of guanidine lipid molecules as a single-component transfection reagent was evaluated.

[0038] Figure 7 The transfection effect of guanidine lipid molecules as a single-component transfection reagent in mice.

[0039] Figure 8 for Figure 7 The quantitative results of fluorescence intensity in the sample. Detailed Implementation

[0040] The present invention will be further described below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention.

[0041] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0042] The compounds involved in the examples are described below: Aminoguanidine hydrochloride, CAS No.: 1937-19-5; 10-Nonadecanone, CAS No.: 504-57-4; 12-Triadecanone, CAS No.: 540-09-0; 14-Heptaconone, CAS No.: 542-50-7; 16-Heptadecanone, CAS No.: 502-73-8; 18-Centaconone, CAS No.: 504-53-0; Boc-glycine, CAS No.: 4530-20-5; Octyldodecyl alcohol, CAS No.: 5333-42-6; Decyltetradecyl alcohol, CAS No.: 58670-89-6; Dodecyl hexadecyl alcohol, CAS No.: 72388-18-2; 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), CAS No.: 7084-11-9; 4-Dimethylaminopyridine (DMAP), CAS No.: 1122-58-3; N,N'-di-BOC-1H-1-guanidinopyrazole, CAS No.: 152120-54-2; (3-Aminopropyl)diethanolamine, CAS No.: 4985-85-7; ditert-butyl dicarbonate, CAS No.: 24424-99-5; triethylamine, CAS No.: 121-44-8; decanoic acid, CAS No.: 334-48-5; dodecanoic acid, CAS No.: 143-07-7; tetradecanoic acid, CAS No.: 544-63-8; hexadecanoic acid, CAS No.: 57-10-3; 2-Butyloctanoic acid, CAS No.: 27610-92-0; 2-Hexyldecanoic acid, CAS No.: 25354-97-6; isostearic acid, CAS No.: 22890-21-7; DSPC, CAS No.: 4539-70-2; Cholesterol, CAS No.: 57-88-5; SM-102, CAS No.: 2089251-47-6; Dlin-MC3-DMA, CAS No.: 1224606-06-7; ALC-0315, CAS No.: 2036272-55-4; DMG-PEG 2kCAS No.: 1397695-86-1; ALC-0519, CAS No.: 1849616-42-7; 1,2-Dioleoyl-3-trimethylammonium-propane (chloride) (DOTAP), CAS No.: 132172-61-3.

[0043] The mRNA encoding green fluorescent protein (GFP) and the mRNA encoding luciferase were purchased from Kaituo Biotechnology.

[0044] Example 1: Synthesis of compounds A1-A5 1. Synthesis of compound A1 10-Ninedecanone (1 g, 3.53 mmol) and aminoguanidine hydrochloride (0.59 g, 5.3 mmol) were dissolved in 10 mL of ethanol. 200 μL of hydrochloric acid was added, and the mixture was heated to reflux at 70 °C for 6 h. The ethanol solvent was then removed by rotary evaporation. 20 mL of DCM was added, and the mixture was stirred to dissolve. The mixture was extracted three times with 100 mL of water, and the organic phase was collected. The organic solvent was then evaporated by rotary evaporation to obtain the crude product. The crude product was purified by column chromatography (silica gel column, eluent: DCM:MeOH = 20:1) to obtain a white powder.

[0045] The 1H NMR spectrum of compound A1 is as follows: 1 H NMR (600 MHz, MeOD) δ 2.32 (q, J =8.3 Hz, 2H), 1.58 (t, J = 7.4 Hz, 1H), 1.52 (t, J = 7.8 Hz, 1H), 1.41 – 1.29(m, 13H), 0.90 (t, J = 6.8 Hz, 3H).

[0046] The structural formula of compound A1 is as follows: .

[0047] 2. Synthesis of compound A2 12-Trichoderma (1 g, 2.9 mmol) and aminoguanidine hydrochloride (0.49 g, 4.4 mmol) were dissolved in 10 mL of ethanol. 200 μL of hydrochloric acid was added, and the mixture was heated to reflux at 70 °C for 6 h. The ethanol solvent was then removed by rotary evaporation. 20 mL of DCM was added, and the mixture was stirred to dissolve. The mixture was extracted three times with 100 mL of water, and the organic phase was collected. The organic solvent was then evaporated by rotary evaporation to obtain the crude product. The crude product was purified by column chromatography (silica gel column, eluent: DCM:MeOH = 20:1) to obtain a yellow solid.

[0048] The 1H NMR spectrum data of compound A2 are as follows: 1H NMR (600 MHz, MeOD) δ 2.33 (q, J = 8.0Hz, 2H), 1.59 (t, J = 7.4 Hz, 1H), 1.52 (t, J = 7.9 Hz, 1H), 1.35 (s, 2H),1.34 (s, 1H), 1.31 (dd, J = 17.6, 5.5 Hz, 11H), 0.90 (t, J = 6.9 Hz, 3H).

[0049] The structural formula of compound A2 is as follows: .

[0050] 3. Synthesis of compound A3 14-Heptaconitone (1 g, 2.5 mmol) and aminoguanidine hydrochloride (0.42 g, 3.8 mmol) were dissolved in 10 mL of ethanol. 200 μL of hydrochloric acid was added, and the mixture was heated to reflux at 70 °C for 6 h. The ethanol solvent was then removed by rotary evaporation. 20 mL of DCM was added, and the mixture was stirred to dissolve. The mixture was extracted three times with 100 mL of water, and the organic phase was collected. The organic solvent was then evaporated by rotary evaporation to obtain the crude product. The crude product was purified by column chromatography (silica gel column, eluent: DCM:MeOH = 20:1) to obtain a yellow solid.

[0051] The 1H NMR spectrum data of compound A3 are as follows: 1 H NMR (600 MHz, MeOD) δ 2.35 – 2.28 (m,2H), 1.57 (q, J = 7.2 Hz, 1H), 1.51 (q, J = 7.8 Hz, 1H), 1.41 – 1.36 (m, 0H), 1.35 (s, 2H), 1.34 (d, J = 4.2 Hz, 1H), 1.31 (d, J = 9.1 Hz, 5H), 1.29 (s,12H), 0.90 (t, J = 6.9 Hz, 3H), 0.87 (s, 1H).

[0052] The structural formula of compound A3 is as follows: .

[0053] 4. Synthesis of compound A4 16-Hexadecanetanone (1 g, 2.2 mmol) and aminoguanidine hydrochloride (0.37 g, 3.3 mmol) were dissolved in 10 mL of ethanol. 200 μL of hydrochloric acid was added, and the mixture was heated to reflux at 70 °C for 6 h. The ethanol solvent was then removed by rotary evaporation. 20 mL of DCM was added, and the mixture was stirred to dissolve. The mixture was extracted three times with 100 mL of water, and the organic phase was collected. The organic solvent was then evaporated by rotary evaporation to obtain the crude product. The crude product was purified by column chromatography (silica gel column, eluent: DCM:MeOH = 20:1) to obtain a white powder.

[0054] 1H NMR data for compound A4: 1 H NMR (400 MHz, MeOD) δ 4.95 (s, 1H), 2.36 –2.27 (m, 1H), 1.55 (dt, J = 22.7, 6.1 Hz, 1H), 1.32 (d, J = 11.4 Hz, 5H), 1.29 (s, 6H), 0.94 – 0.86 (m, 1H), 0.77 (s, 1H).

[0055] The structural formula of compound A4 is as follows: .

[0056] 5. Synthesis of compound A5 18-Trispentacosanone (1 g, 2.0 mmol) and aminoguanidine hydrochloride (0.33 g, 3.0 mmol) were dissolved in 10 mL of ethanol. 200 μL of hydrochloric acid was added, and the mixture was heated to reflux at 70 °C for 6 h. The ethanol solvent was then removed by rotary evaporation. 20 mL of DCM was added, and the mixture was stirred to dissolve. The mixture was extracted three times with 100 mL of water, and the organic phase was collected. The organic solvent was then evaporated by rotary evaporation to obtain the crude product. The crude product was purified by column chromatography (silica gel column, eluent: DCM:MeOH = 20:1) to obtain a white powder.

[0057] 1H NMR data for compound A5: 1H NMR (600 MHz, MeOD) δ 2.32 (td, J = 9.3,7.2 Hz, 2H), 1.59 (p, J = 7.0 Hz, 1H), 1.53 (t, J = 7.8 Hz, 1H), 1.37 – 1.32(m, 4H), 1.31 (d, J = 7.1 Hz, 1H), 1.31 (s, 5H), 1.29 (s, 12H), 1.27 (s, 1H), 1.18 (s, 0H), 0.90 (t, J = 7.0 Hz, 3H).

[0058] The structural formula of compound A5 is as follows: .

[0059] Example 2: Synthesis of compounds B1-B3 1. Synthesis of compound B1 The synthesis route is as follows:

[0060] Boc-glycine (2 g, 11.4 mmol), octyldodecyl alcohol (8.54 g, 28.6 mmol), EDCI (5.49 g, 28.6 mmol), and DMAP (0.56 g, 4.6 mmol) were dissolved in 20 mL of dichloromethane and stirred at room temperature for 12 h. The product was purified by column chromatography with hexane:ethyl acetate as the eluent to obtain product B1-1. B1-1 was dissolved in 3 mL of dichloromethane, and 1 mL of trifluoroacetic acid (CF3COOH) was added under ice bath conditions. The reaction was carried out for two hours to obtain the crude product. The crude product was extracted successively with saturated sodium bicarbonate solution, pure water, and saturated brine to remove trifluoroacetic acid, yielding product B1-2.

[0061] B1-2 (0.5 g, 1.2 mmol) and N,N'-di-BOC-1H-1-guanidinopyrazole (0.56 g, 1.8 mmol) were dissolved in 10 mL of dichloromethane, and 0.5 mL of triethylamine was added. The mixture was stirred at room temperature for 12 h. The product was purified by column chromatography using n-hexane:ethyl acetate as the eluent to obtain product B1-3. B1-3 was dissolved in 1.5 mL of dichloromethane, and 0.5 mL of CF3COOH was added under ice bath conditions. The reaction was carried out for two hours to obtain the crude product. The crude product was extracted successively with saturated sodium bicarbonate solution, pure water, and saturated brine to remove trifluoroacetic acid, yielding product B1.

[0062] 1H NMR data for compound B1: 1H NMR (600 MHz, CDCl3) δ 4.05 (d, J = 5.8 Hz, 2H), 3.97 (d, J = 5.4 Hz, 2H), 1.69 – 1.58 (m, 1H), 1.26 (s, 32H), 0.88 (t, J= 7.0 Hz, 6H).

[0063] The structural formula of compound B1 is as follows: .

[0064] 2. Synthesis of compound B2 The synthetic route was similar to that of B1. Boc-glycine (2 g, 11.4 mmol), decyltetradecyl alcohol (10.15 g, 28.6 mmol), EDCI (5.49 g, 28.6 mmol), and DMAP (0.56 g, 4.6 mmol) were dissolved in 20 mL of dichloromethane and stirred at room temperature for 12 h. The product was purified by column chromatography using n-hexane:ethyl acetate as the eluent to obtain product B2-1. B2-1 was dissolved in 3 mL of dichloromethane, and 1 mL of trifluoroacetic acid (CF3COOH) was added under ice bath conditions. The reaction was carried out for two hours to obtain the crude product. The crude product was extracted successively with saturated sodium bicarbonate solution, pure water, and saturated brine to remove trifluoroacetic acid, yielding product B2-2.

[0065] B2-2 (0.5 g, 1.2 mmol) and N,N'-di-BOC-1H-1-guanidinopyrazole (0.65 g, 2.1 mmol) were dissolved in 10 mL of dichloromethane, and 0.58 mL of triethylamine was added. The mixture was stirred at room temperature for 12 h. The product was purified by column chromatography with hexane:ethyl acetate as the eluent in a 5:1 ratio to obtain product B2-3. B2-3 was dissolved in 1.5 mL of dichloromethane, and 0.5 mL of CF3COOH was added under ice bath conditions. The reaction was carried out for two hours to obtain the crude product. The crude product was extracted successively with saturated sodium bicarbonate solution, pure water, and saturated brine to remove trifluoroacetic acid, yielding product B2.

[0066] 1H NMR data for compound B2: 1 H NMR (600 MHz, MeOD) δ 4.11 (d, J = 5.7 Hz, 2H), 4.05 (s, 2H), 1.68 (s, 1H), 1.32 (dd, J = 24.7, 5.5 Hz, 40H), 0.90 (t, J= 6.9 Hz, 6H). The structural formula of compound B2 is as follows: .

[0067] 3. Synthesis of compound B3 The synthetic route was similar to that of B1. Boc-glycine (2 g, 11.4 mmol), dodecyl hexadecyl alcohol (11.75 g, 28.6 mmol), EDCI (5.49 g, 28.6 mmol), and DMAP (0.56 g, 4.6 mmol) were dissolved in 20 mL of dichloromethane and stirred at room temperature for 12 h. The product was purified by column chromatography using n-hexane:ethyl acetate as the eluent to obtain product B3-1. B3-1 was dissolved in 3 mL of dichloromethane, and 1 mL of trifluoroacetic acid (CF3COOH) was added under ice bath conditions. The reaction was carried out for two hours to obtain the crude product. The crude product was extracted successively with saturated sodium bicarbonate solution, pure water, and saturated brine to remove trifluoroacetic acid, yielding product B3-2.

[0068] B3-2 (0.5 g, 1.1 mmol) and N,N'-di-BOC-1H-1-guanidinopyrazole (0.51 g, 1.65 mmol) were dissolved in 10 mL of dichloromethane, and 0.46 mL of triethylamine was added. The mixture was stirred at room temperature for 12 h. The product was purified by column chromatography using n-hexane:ethyl acetate as the eluent to obtain product B3-3. B3-3 was dissolved in 1.5 mL of dichloromethane, and 0.5 mL of CF3COOH was added under ice bath conditions. The reaction was carried out for two hours to obtain the crude product. The crude product was extracted successively with saturated sodium bicarbonate solution, pure water, and saturated brine to remove trifluoroacetic acid, yielding product B3.

[0069] 1H NMR data for compound B3: 1 H NMR (600 MHz, MeOD) δ 4.11 (d, J = 5.7 Hz, 2H), 4.05 (s, 2H), 1.72 – 1.65 (m, 1H), 1.29 (d, J = 6.7 Hz, 48H), 0.89 (q, J= 7.0 Hz, 6H). The structural formula of compound B3 is as follows: .

[0070] Example 3: Synthesis of compounds C1-C4 1. Synthesis of compound C1 The synthesis route is as follows:

[0071] (3-Aminopropyl)diethanolamine (2 g, 12.3 mmol) was dissolved in 10 mL of dichloroethane. Ditert-butyl dicarbonate (5.38 g, 24.7 mmol) and triethylamine (3.43 mL, 24.7 mmol) were added under ice bath conditions. The mixture was stirred overnight, washed three times with water, and the organic solvent was removed by rotary evaporation to obtain intermediate product M.

[0072] M (1 g, 3.8 mmol), decanoic acid (1.63 g, 9.5 mmol), EDCI (1.82 g, 9.5 mmol), and DMAP (0.18 g, 1.5 mmol) were dissolved in 20 mL of dichloromethane and stirred at room temperature for 12 h. The product was purified by column chromatography with hexane:ethyl acetate as the eluent to give product C1-1. D3-1 was dissolved in 3 mL of dichloromethane, and 1 mL of CF3COOH was added under ice bath conditions. The reaction was carried out for two hours to obtain the crude product. The crude product was extracted successively with saturated sodium bicarbonate solution, pure water, and saturated brine to remove trifluoroacetic acid, giving product C1-2.

[0073] C1-2 (0.5 g, 1.06 mmol) and N,N'-di-BOC-1H-1-guanidinopyrazole (0.49 g, 1.59 mmol) were dissolved in 10 mL of dichloromethane, and 0.36 mL of triethylamine was added. The mixture was stirred at room temperature for 12 h. The product was purified by column chromatography with dichloromethane:methanol = 50 as the eluent to obtain product C1-3. C1-3 was dissolved in 1.5 mL of dichloromethane, and 0.5 mL of CF3COOH was added under ice bath conditions. The reaction was carried out for two hours to obtain the crude product. The crude product was extracted successively with saturated sodium bicarbonate solution, pure water, and saturated brine to remove trifluoroacetic acid, yielding product C1.

[0074] 1H NMR data for compound C1: 1 H NMR (600 MHz, CDCl3) δ 4.44 (t, J = 4.8 Hz, 4H), 3.51 (t, J = 4.9 Hz, 4H), 3.35 (p, J = 7.7, 7.3 Hz, 4H), 2.33 (t, J =7.7 Hz, 4H), 2.10 (q, J = 7.5, 7.0 Hz, 2H), 1.59 (q, J = 7.2 Hz, 4H), 1.27 (d, J = 12.1 Hz, 24H), 0.87 (t, J = 7.0 Hz, 6H). The structural formula of compound C1 is as follows: .

[0075] 2. Synthesis of compound C2 The synthetic route was similar to that of C1. M (1 g, 3.8 mmol), dodecanoic acid (1.9 g, 9.5 mmol), EDCI (1.82 g, 9.5 mmol), and DMAP (0.18 g, 1.5 mmol) were dissolved in 20 mL of dichloromethane and stirred at room temperature for 12 h. The product was purified by column chromatography with hexane:ethyl acetate as the eluent to obtain product C2-1. C2-1 was dissolved in 3 mL of dichloromethane, and 1 mL of CF3COOH was added under ice bath conditions. The reaction was carried out for two hours to obtain the crude product. The crude product was extracted successively with saturated sodium bicarbonate solution, pure water, and saturated brine to remove trifluoroacetic acid, yielding product C2-2.

[0076] C2-2 (0.5 g, 0.95 mmol) and N,N'-di-BOC-1H-1-guanidinopyrazole (0.44 g, 1.43 mmol) were dissolved in 10 mL of dichloromethane, and 0.39 mL of triethylamine was added. The mixture was stirred at room temperature for 12 h. The product was purified by column chromatography with dichloromethane:methanol = 50 as the eluent to obtain product C2-3. C2-3 was dissolved in 1.5 mL of dichloromethane, and 0.5 mL of CF3COOH was added under ice bath conditions. The reaction was carried out for two hours to obtain the crude product. The crude product was extracted successively with saturated sodium bicarbonate solution, pure water, and saturated brine to remove trifluoroacetic acid, yielding product C2.

[0077] 1H NMR data for compound C2: 1 H NMR (600 MHz, CDCl3) δ 4.16 (t, J = 5.8 Hz,4H), 3.24 (t, J = 6.7 Hz, 2H), 2.79 (t, J = 5.8 Hz, 4H), 2.63 (t, J = 6.6 Hz,2H), 2.32 (q, J = 8.8, 8.1 Hz, 4H), 1.74 (q, J = 6.6 Hz, 2H), 1.60 (q, J =7.4 Hz, 4H), 1.30 (d, J = 11.5 Hz, 32H), 0.90 (t, J = 6.8 Hz, 6H). The structural formula of compound C2 is as follows: .

[0078] 3. Synthesis of compound C3 The synthetic route was similar to that of C1. M (1 g, 3.8 mmol), myristic acid (2.2 g, 9.5 mmol), EDCI (1.82 g, 9.5 mmol), and DMAP (0.18 g, 1.5 mmol) were dissolved in 20 mL of dichloromethane and stirred at room temperature for 12 h. The product was purified by column chromatography with hexane:ethyl acetate as the eluent to obtain product C3-1. C3-1 was dissolved in 3 mL of dichloromethane, and 1 mL of CF3COOH was added under ice bath conditions. The reaction was allowed to proceed for two hours to obtain the crude product. The crude product was then extracted successively with saturated sodium bicarbonate solution, pure water, and saturated brine to remove trifluoroacetic acid, yielding product C3-2.

[0079] C3-2 (0.5 g, 0.86 mmol) and N,N'-di-BOC-1H-1-guanidinopyrazole (0.40 g, 1.29 mmol) were dissolved in 10 mL of dichloromethane, and 0.36 mL of triethylamine was added. The mixture was stirred at room temperature for 12 h. The product was purified by column chromatography with dichloromethane:methanol = 50 as the eluent to obtain product C3-3. C3-3 was dissolved in 1.5 mL of dichloromethane, and 0.5 mL of CF3COOH was added under ice bath conditions. The reaction was carried out for two hours to obtain the crude product. The crude product was extracted successively with saturated sodium bicarbonate solution, pure water, and saturated brine to remove trifluoroacetic acid, yielding product C3.

[0080] 1H NMR data for compound C3: 1 H NMR (600 MHz, CDCl3) δ 4.44 (t, J = 5.2 Hz, 4H), 3.50 (t, J = 5.1 Hz, 4H), 3.34 (p, J = 6.6, 5.9 Hz, 4H), 2.33 (t, J =7.7 Hz, 4H), 2.14 – 2.06 (m, 2H), 1.59 (t, J = 7.3 Hz, 4H), 1.36 – 1.16 (m, 40H), 0.88 (t, J = 7.0 Hz, 6H). The structural formula of compound C3 is as follows: .

[0081] 4. Synthesis of compound C4 The synthetic route was similar to that of C1. M (1 g, 3.8 mmol), hexadecanoic acid (2.4 g, 9.5 mmol), EDCI (1.82 g, 9.5 mmol), and DMAP (0.18 g, 1.5 mmol) were dissolved in 20 mL of dichloromethane and stirred at room temperature for 12 h. The product was purified by column chromatography with hexane:ethyl acetate as the eluent to obtain product C4-1. C4-1 was dissolved in 3 mL of dichloromethane, and 1 mL of CF3COOH was added under ice bath conditions. The reaction was carried out for two hours to obtain the crude product. The crude product was extracted successively with saturated sodium bicarbonate solution, pure water, and saturated brine to remove trifluoroacetic acid, yielding product C4-2.

[0082] C4-2 (0.5 g, 0.78 mmol) and N,N'-di-BOC-1H-1-guanidinopyrazole (0.36 g, 1.17 mmol) were dissolved in 10 mL of dichloromethane, and 0.32 mL of triethylamine was added. The mixture was stirred at room temperature for 12 h. The product was purified by column chromatography with dichloromethane:methanol = 50 as the eluent to obtain product C4-3. C4-3 was dissolved in 1.5 mL of dichloromethane, and 0.5 mL of CF3COOH was added under ice bath conditions. The reaction was carried out for two hours to obtain the crude product. The crude product was extracted successively with saturated sodium bicarbonate solution, pure water, and saturated brine to remove trifluoroacetic acid, yielding product C4.

[0083] 1H NMR data for compound C4: 1 H NMR (600 MHz, CDCl3) δ 4.16 (t, J = 5.7 Hz, 4H), 3.29 (dt, J = 24.9, 6.2 Hz, 2H), 2.82 – 2.73 (m, 4H), 2.62 (t, J = 5.9Hz, 2H), 2.30 (q, J = 7.9 Hz, 4H), 1.76 (p, J = 6.0 Hz, 2H), 1.60 (q, J = 7.1Hz, 4H), 1.26 (s, 48H), 0.88 (t, J = 7.0 Hz, 6H). The structural formula of compound C4 is as follows: .

[0084] Example 4: Synthesis of compounds D1-D3 1. Synthesis of compound D1 The synthesis route is as follows:

[0085] M (1 g, 3.8 mmol), 2-butyloctanoic acid (1.9 g, 9.5 mmol), EDCI (1.82 g, 9.5 mmol), and DMAP (0.18 g, 1.5 mmol) were dissolved in 20 mL of dichloromethane and stirred at room temperature for 12 h. The product was purified by column chromatography with hexane:ethyl acetate as the eluent to obtain product D1-1. D1-1 was dissolved in 3 mL of dichloromethane, and 1 mL of CF3COOH was added under ice bath conditions. The reaction was carried out for two hours to obtain the crude product. The crude product was extracted successively with saturated sodium bicarbonate solution, pure water, and saturated brine to remove trifluoroacetic acid, yielding product D1-2.

[0086] D1-2 (0.5 g, 0.95 mmol) and N,N'-di-BOC-1H-1-guanidinopyrazole (0.44 g, 1.43 mmol) were dissolved in 10 mL of dichloromethane, and 0.39 mL of triethylamine was added. The product was purified by column chromatography with dichloromethane:methanol = 50 as the eluent to obtain product D1-3. D1-3 was dissolved in 1.5 mL of dichloromethane, and 0.5 mL of CF3COOH was added under ice bath conditions. The reaction was carried out for two hours to obtain the crude product. The crude product was extracted successively with saturated sodium bicarbonate solution, pure water, and saturated brine to remove trifluoroacetic acid, yielding product D1.

[0087] 1H NMR data for compound 12: 1 H NMR (600 MHz, CDCl3) δ 4.18 (t, J = 6.0 Hz, 4H), 3.25 (q, J = 6.2 Hz, 2H), 2.82 (q, J = 5.9, 5.3 Hz, 4H), 2.64 (t, J = 6.1 Hz, 2H), 2.33 (td, J = 8.8, 4.4 Hz, 2H), 1.78 (p, J = 6.1 Hz, 2H), 1.58 (dq, J = 15.6,8.4 Hz, 4H), 1.45 (ddt, J = 14.6, 9.7, 5.4 Hz, 4H), 1.37 – 1.09 (m, 32H), 0.88(td, J = 7.1, 3.7 Hz, 12H). The structural formula of compound D1 is as follows: .

[0088] 2. Synthesis of compound D2 The synthetic route was similar to that of D1. M (1 g, 3.8 mmol), 2-hexyldecanoic acid (2.43 g, 9.5 mmol), EDCI (1.82 g, 9.5 mmol), and DMAP (0.18 g, 1.5 mmol) were dissolved in 20 mL of dichloromethane and stirred at room temperature for 12 h. The product was purified by column chromatography with hexane:ethyl acetate as the eluent to obtain product D2-1. D2-1 was dissolved in 3 mL of dichloromethane, and 1 mL of CF3COOH was added under ice bath conditions. The reaction was carried out for two hours to obtain the crude product. The crude product was extracted successively with saturated sodium bicarbonate solution, pure water, and saturated brine to remove trifluoroacetic acid, yielding product D2-2.

[0089] D2-2 (0.5 g, 0.78 mmol) and N,N'-di-BOC-1H-1-guanidinopyrazole (0.36 g, 1.17 mmol) were dissolved in 10 mL of dichloromethane, and 0.32 mL of triethylamine was added. The mixture was stirred at room temperature for 12 h. The product was purified by column chromatography with dichloromethane:methanol = 50 as the eluent to obtain product D2-3. D2-3 was dissolved in 1.5 mL of dichloromethane, and 0.5 mL of CF3COOH was added under ice bath conditions. The reaction was carried out for two hours to obtain the crude product. The crude product was extracted successively with saturated sodium bicarbonate solution, pure water, and saturated brine to remove trifluoroacetic acid, yielding product D2.

[0090] 1H NMR data for compound D2: 1 H NMR (600 MHz, CDCl3) δ 4.17 (t, J = 6.0 Hz,4H), 3.21 (d, J = 7.2 Hz, 2H), 2.80 (t, J = 6.0 Hz, 4H), 2.63 (t, J = 6.1 Hz,2H), 2.32 (tt, J = 8.6, 5.5 Hz, 2H), 1.75 (p, J = 6.4 Hz, 2H), 1.56 (q, J =8.3, 7.8 Hz, 4H), 1.45 (td, J = 16.3, 13.6, 10.3 Hz, 4H), 1.34 – 1.18 (m,40H), 0.88 (t, J = 6.8 Hz, 12H). The structural formula of compound D2 is as follows: .

[0091] 3. Synthesis of compound D3 The synthetic route was similar to that of D1. M (1 g, 3.8 mmol), isostearic acid (2.70 g, 9.5 mmol), EDCI (1.82 g, 9.5 mmol), and DMAP (0.18 g, 1.5 mmol) were dissolved in 20 mL of dichloromethane and stirred at room temperature for 12 h. The product was purified by column chromatography with hexane:ethyl acetate as the eluent to obtain product D3-1. D3-1 was dissolved in 3 mL of dichloromethane, and 1 mL of CF3COOH was added under ice bath conditions. The reaction was allowed to proceed for two hours to obtain the crude product. The crude product was then extracted successively with saturated sodium bicarbonate solution, pure water, and saturated brine to remove trifluoroacetic acid, yielding product D3-2.

[0092] D3-2 (0.5 g, 0.72 mmol) and N,N'-di-BOC-1H-1-guanidinopyrazole (0.33 g, 1.08 mmol) were dissolved in 10 mL of dichloromethane, and 0.3 mL of triethylamine was added. The mixture was stirred at room temperature for 12 h. The product was purified by column chromatography with dichloromethane:methanol = 50 as the eluent to obtain product D3-3. D3-3 was dissolved in 1.5 mL of dichloromethane, and 0.5 mL of CF3COOH was added under ice bath conditions. The reaction was carried out for two hours to obtain the crude product. The crude product was extracted successively with saturated sodium bicarbonate solution, pure water, and saturated brine to remove trifluoroacetic acid, yielding product D3.

[0093] 1H NMR data for compound D3: 1 H NMR (600 MHz, CDCl3) δ 4.16 (t, J = 5.6 Hz, 4H), 3.26 (q, J = 6.1 Hz, 2H), 2.79 (t, J = 5.7 Hz, 4H), 2.62 (d, J = 6.1 Hz, 2H), 2.32 (t, J = 7.6 Hz, 4H), 1.77 (q, J = 6.2 Hz, 2H), 1.60 (t, J = 7.1 Hz, 6H), 1.27(d, J = 17.3 Hz, 48H), 0.86 (dt, J = 24.2, 6.7 Hz, 12H). The structural formula of compound D3 is as follows: .

[0094] Application Example 1: Guanidinyl lipid molecules as transfection enhancers promote the delivery of mRNA by traditional four-component lipid nanoparticles. 1. Preparation of five-component lipid nanoparticles containing guanidine lipid molecules The compounds from Examples 1-4 were combined with SM102, DSPC, cholesterol, and DMG-PEG. 2k A lipid ethanol solution was prepared by dissolving the lipid in ethanol at a molar ratio of 5:50:10:38.5:1.5. The mRNA encoding green fluorescent protein (GFP) was dissolved in 20 mM sodium acetate buffer. The lipid and nucleic acid solutions were mixed at a volume ratio of 1:3 and a mass ratio of 40:1, vortexed thoroughly, and dialyzed to remove ethanol molecules, yielding a five-component lipid nanoparticle solution (SM-LNP / X) containing guanidine lipid molecules.

[0095] 2. Particle size characterization of five-component lipid nanoparticles containing guanidine lipid molecules The hydration kinetics and particle size distribution of the five-component lipid nanoparticles were determined using a Malvern particle size analyzer. The experiment was repeated three times and the average value was taken. The data results are shown in Table 1. The results show that the particle size of SM-LNP is between 100-200 nm, the PDI is basically less than 0.25, and the particle size distribution is narrow, which makes it suitable for in vivo and in vitro drug delivery.

[0096] 3. Characterization of the encapsulation efficiency of mRNA encapsulated by five-component lipid nanoparticles containing guanidine lipid molecules. Five-component lipid nanoparticles containing 1 μg mRNA were prepared. The lipid nanoparticles were diluted with TE buffer or TE buffer containing 2% Triton X to detect free mRNA and total mRNA concentrations. A 1 μg / mL mRNA solution was prepared and diluted twofold to 31.25 μg / mL for use in the standard curve detection. 100 μL of the above solution was mixed with 100 μL of Libogreen solution (flash crystal, 1:200 dilution), and the fluorescence intensity was detected using a microplate reader at an excitation wavelength of 485 nm and an emission wavelength of 528 nm. The mRNA encapsulation efficiency was calculated using the following formula, and the results are shown in Table 1.

[0097] .

[0098] Table 1. Characterization of guanidine-based lipid nanoparticles

[0099] 4. Verify at the cellular level the effect of guanidine lipid molecules on enhancing the delivery of mRNA by lipid nanoparticles. Add 2×10 to each well of the 48-well plate 4After 24 hours, lipid nanoparticles containing 0.5 μg GFP-mRNA were added to 200 μL LMEM medium in each well of a 48-well plate, replacing the cell culture medium. GFP expression was observed after 24 hours, and the mean fluorescence intensity (MFI) was calculated using FlowJo software.

[0100] As a control, lipid nanoparticles SM-LNP without guanidine lipid molecules were prepared. SM-102, DSPC, cholesterol, and DMG-PEG were mixed in ethanol as a solvent. 2k Dissolve 0.5 μg of GFP-encoding mRNA in a 20 mM sodium acetate buffer solution. Mix the lipid solution and nucleic acid solution at a volume ratio of 1:3 and a mass ratio of 40:1, vortex thoroughly, and dialyze to remove ethanol molecules to obtain lipid nanoparticles SM-LNP. Add SM-LNP containing 0.5 μg of GFP-mRNA to DMEM medium, replacing the cell culture medium in 48-well plates. Observe GFP expression after 24 h, and calculate the mean fluorescence intensity (MFI) using FlowJo software.

[0101] Cell transfection results as follows Figure 1 As shown, the MFI results are as follows Figure 2 As shown in the figure. The results show that for lipid nanoparticles with different components, the addition of guanidine lipid molecules can increase the expression intensity of mRNA. Among them, A4, A5, B3, C3, C4, D2, and D3 significantly increased the expression intensity of mRNA, indicating that the guanidine lipid molecules can promote the delivery of mRNA by traditional lipid nanoparticles.

[0102] 5. Verify the effect of guanidine lipid molecules on enhancing the delivery of mRNA by lipid nanoparticles in various lipid nanoparticle formulations. Compound A4 was dissolved in ethanol at a molar ratio of 5:46.3:9.4:42.7:1.6 to prepare a lipid ethanol solution. The mRNA encoding green fluorescent protein (GFP) was dissolved in 20 mM sodium acetate buffer. The lipid solution and nucleic acid solution were mixed at a volume ratio of 1:3 and a mass ratio of 40:1, vortexed thoroughly, and dialyzed to remove ethanol molecules, yielding a five-component lipid nanoparticle solution (ALC-LNP / A4) containing guanidine lipid molecules.

[0103] Compound A4 was combined with MC3, DSPC, cholesterol, and DMG-PEG. 2kA lipid ethanol solution was prepared by dissolving the lipid in ethanol at a molar ratio of 5:50:10:38.5:1.5. The mRNA encoding green fluorescent protein (GFP) was dissolved in 20 mM sodium acetate buffer. The lipid and nucleic acid solutions were mixed at a volume ratio of 1:3 and a mass ratio of 40:1, vortexed thoroughly, and dialyzed to remove ethanol molecules, yielding a five-component lipid nanoparticle solution (MC3-LNP / A4) containing guanidine lipid molecules.

[0104] Simultaneously, a control group was prepared by dissolving ALC-0315, DSPC, cholesterol, and ALC-0519 in ethanol at a molar ratio of 46.3:9.4:42.7:1.6. The mRNA encoding GFP was dissolved in 20 mM sodium acetate buffer solution. The lipid solution and nucleic acid solution were mixed at a volume ratio of 1:3 and a mass ratio of 40:1, vortexed thoroughly, and dialyzed to remove ethanol molecules, yielding lipid nanoparticles ALC-LNP.

[0105] Using ethanol as a solvent, MC3, DSPC, cholesterol, and DMG-PEG were combined. 2k The GFP-encoding mRNA was dissolved in a molar ratio of 50:10:38.5:1.5. The lipid and nucleic acid solutions were then mixed at a volume ratio of 1:3 and a mass ratio of 40:1, vortexed thoroughly, and dialyzed to remove ethanol molecules, yielding lipid nanoparticles MC3-LNP.

[0106] Add 5×10 to each well of the 24-well plate 4 BHK cells were cultured, and after 24 hours, the lipid nanoparticles were added to 500 μL of culture medium, replacing the cell culture medium in the 24-well plates. GFP expression was observed after 24 hours, and flow cytometry was used to analyze GFP expression in the cells.

[0107] Cell transfection rate and fluorescence expression intensity results are as follows: Figure 3 As shown in the figure. The results showed that adding 5% A4 to both MC3-LNP and ALC-LNP formulations significantly improved cell transfection efficiency.

[0108] 6. Validating the effect of guanidine lipid molecules on enhancing the delivery of mRNA by lipid nanoparticles in multiple cell models. Add 5×10 to each well of the 24-well plate. 4BHK cells, 4T1 cells, and 3T3 cells were cultured. After 24 hours, lipid nanoparticles SM-LNP / A4 containing 1 μg GFP-mRNA were added to 500 μL of culture medium, replacing the cell culture medium in the 24-well plates. The control group was prepared by adding SM-LNP containing 1 μg GFP-mRNA to the culture medium in the same manner. GFP expression was observed after 24 hours, and the GFP expression level in the cells was analyzed using flow cytometry.

[0109] Cell transfection rate and fluorescence expression intensity results are as follows: Figure 4 As shown in the figure. The results showed that, in various cell models, cells treated with lipid nanoparticles containing A4 (SM-LNP / A4) had stronger mRNA entry efficiency and expression intensity compared with the four-component lipid nanoparticles, indicating that compound A4 can play a role in promoting the delivery of mRNA and transfection by lipid nanoparticles in various cell models.

[0110] 7. Validating the effect of guanidine lipid molecules on enhancing the delivery of mRNA by lipid nanoparticles at the animal level. First, lipid nanoparticles containing guanidine-containing lipid molecules loaded with Luci-mRNA were prepared. Compound A4 was then combined with SM102, DSPC, cholesterol, and DMG-PEG. 2k A lipid ethanol solution was prepared by dissolving the lipid in ethanol at a molar ratio of 5:50:10:38.5:1.5. The mRNA encoding firefly luciferase (Luci) was dissolved in 20 mM sodium acetate buffer. The lipid and nucleic acid solutions were mixed at a volume ratio of 1:3 and a mass ratio of 40:1, vortexed thoroughly, and dialyzed to remove ethanol molecules, yielding a five-component lipid nanoparticle solution (SM-LNP / A4 / Luci) containing guanidine lipid molecules.

[0111] Simultaneously, a control group was prepared, in which SM-102, DSPC, cholesterol, and DMG-PEG were mixed using ethanol as a solvent. 2k The mRNA encoding firefly luciferase (Luci) was dissolved in a 20 mM sodium acetate buffer solution at a molar ratio of 50:10:38.5:1.5. The lipid solution and nucleic acid solution were mixed at a volume ratio of 1:3 and a mass ratio of 40:1, vortexed thoroughly, and dialyzed to remove ethanol molecules, thus preparing lipid nanoparticles SM-LNP / Luci.

[0112] Balb / C mice aged 4-6 weeks were randomly divided into two groups of 3 mice each. SM-LNP / A4 / Luci and SM-LNP / Luci were injected intramuscularly into the hind leg muscles of each mouse, with each mouse receiving 2 μg of Luci-mRNA. Four hours post-injection, in vivo luciferase expression was observed using small animal in vivo imaging and quantitatively analyzed using fluorescence.

[0113] In vivo transfection results as follows Figure 5 As shown in the figure. The results showed that after the lipid nanoparticles with added A4 were injected into mice, there was stronger luciferase expression in the mouse muscles, indicating that at the animal level, the guanidinolipid molecule A4 can enhance the delivery of mRNA and transfection effects of lipid nanoparticles.

[0114] Application Example 2: Guanidinyl lipid molecules as a single-component transfection reagent for transfecting mRNA into cells. 1. Verify the effectiveness of guanidine lipid molecules as a single-component transfection reagent for mRNA transfection at the cellular level. Add 5×10 to each well of the 24-well plate 4 HeLa cells were used. After 24 h, 0.3 μg of GFP-encoding mRNA was diluted with 20 mM sodium acetate buffer and mixed with ethanol solutions of the compounds from Examples 1-4. The volume ratio of lipid solution to nucleic acid solution was 1:3, and the molar ratio of lipid to nucleic acid was 5. After mixing, the mixture was vortexed thoroughly, incubated for 10 min, and then DMEM complete medium was added to replace the medium in the 24-well plates. The control material was the cationic lipid transfection reagent DOTAP. GFP expression was observed after 24 h, and the GFP expression in the cells was analyzed using flow cytometry.

[0115] Cell transfection rate and fluorescence expression intensity, such as Figure 6 As shown in the figure. The results showed that molecules A2, C4, and D2 had higher cell transfection rates and stronger green fluorescent protein expression compared to DOTAP, indicating that these lipid molecules, when combined with mRNA, can achieve intracellular transfection of mRNA, and the effect is better than that of the cationic lipid transfection reagent DOTAP.

[0116] 2. Validating the effectiveness of guanidinolipid molecules as a single-component transfection reagent for mRNA transfection at the animal level. Dilute 2 μg of luciferase-encoding mRNA (mLuci) with 20 mM sodium acetate buffer and mix with ethanol solutions of the compounds from Examples 1-4. The volume ratio of lipid solution to nucleic acid solution is 1:3, and the molar ratio of lipid to nucleic acid is 5. Vortex thoroughly after mixing, incubate for 10 min, dilute with 100 μL PBS, and inject via tail vein into 4-6 week old Balb / C mice. Six h post-injection, observe in vivo luciferase expression using small animal in vivo imaging and perform quantitative fluorescence analysis.

[0117] In vivo transfection results as follows Figure 7 and Figure 8 As shown in the figure. The results indicate that guanidinolipid molecules can transfect mRNA in mice, with A2, A3, B3, and D2 showing stronger transfection effects in vivo.

[0118] In summary, this invention provides a series of guanidine-based lipid molecules, each composed of a guanidine head and a fatty acid tail, connected by hydrazone or ester bonds. These guanidine-based lipid molecules have two applications: firstly, they can be added in small amounts as transfection enhancers to four-component lipid nanoparticles to improve the transfection efficacy of mRNA-loaded lipid nanoparticles both in vivo and in vitro. Secondly, they can be used as transfection reagents to directly bind to mRNA molecules, achieving intracellular transfection. Both applications have been experimentally verified, resulting in improved transfection efficiency.

Claims

1. A guanidine lipid compound, characterized in that, The structural formula of the compound is shown in any of formulas (I)-(IV). (AND), (II)、 (III)、 (IV)? R1 and R2 are alkyl chains with 4-17 carbon atoms, respectively.

2. The guanidine lipid compound according to claim 1, characterized in that, R1 and R2 are alkyl chains with 7-17 carbon atoms.

3. The guanidine lipid compound according to claim 1 or 2, characterized in that, The structural formula of the compound is shown in any one of formulas (A1) to (A5), (B1) to (B3), (C1) to (C4), and (D1) to (D3). (A1)； (A2)； (A3)； (A4)； (A5)； (B1)； (B2)； (B3)； (C1)； (C2)； (C3)； (C4)； (D1)； (D2)； (D3)。 4. A lipid nanoparticle, characterized in that, The lipid nanoparticles comprise the guanidine lipid compound as described in any one of claims 1-3, ionizable lipids, phospholipids, cholesterol, and lipids containing polyethylene glycol.

5. The lipid nanoparticles as described in claim 4, characterized in that, The lipid nanoparticles contain guanidine lipid compounds in a molar amount that accounts for 5-10% of the total molar amount of ionizable lipids, phospholipids, cholesterol, and lipids containing polyethylene glycol; the molar ratio of ionizable lipids, phospholipids, cholesterol, and lipids containing polyethylene glycol is 45-50:8-10:35-45:0.5-2.

6. The use of the lipid nanoparticles as described in claim 4 or 5 as a carrier in the preparation of nucleic acid delivery drugs.

7. The application as described in claim 6, characterized in that, The application includes: adding each component to an acidic buffer containing nucleic acid, self-assembling to form lipid nanoparticles carrying nucleic acid, and preparing the nucleic acid delivery drug.

8. The use of the guanidine lipid compound as a carrier in the preparation of nucleic acid delivery drugs according to any one of claims 1-3.

9. The application as described in claim 8, characterized in that, The application includes: adding the guanidine lipid compound to an acidic buffer containing nucleic acid to form a lipid / nucleic acid complex, thereby preparing the nucleic acid delivery drug.

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