Preparation and application of lung targeting LNP based on cationic lipopeptide

Abstract

The invention provides a lung targeting lipid nanoparticle (LNP) based on cationic lipopeptide as well as a preparation method and application of the lung targeting lipid nanoparticle. The lung targeting LNP is composed of cationic lipopeptide, DLin MC3-DMA, cholesterol and DMG-PEG 2000, and nucleic acid can be efficiently encapsulated and compressed through electrostatic interaction, so that high nucleic acid encapsulation efficiency is obtained. Based on the positive charges carried by the cationic lipopeptide, the LNP can adsorb specific biomolecules in plasma to form a protein crown structure, and the lung cell endocytosis of the LNP is promoted by utilizing the corona effect, so that efficient active targeting on the lung is realized. The LNP provided by the invention can specifically deliver nucleic acid to the lung, supports stable gene expression, and is suitable for treating related diseases of the lung. Compared with an existing SORT LNP technology, a large amount of cationic lipopeptide does not need to be additionally added, the total lipid dosage is low, and better biocompatibility and safety are shown.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a lung-targeting LNP based on cationic lipopeptides, as well as the preparation method and application of the above-mentioned lung-targeting LNP. Background Technology

[0002] Lipid nanoparticles (LNPs) are typically composed of ionizable lipids, auxiliary lipids, cholesterol, and polyethylene glycol (PEG) lipids, each playing a crucial role in the delivery process. Ionizable lipids, protonated under acidic conditions, facilitate efficient encapsulation of nucleic acid drugs and promote endosome escape after cell entry. Auxiliary lipids and cholesterol stabilize the LNP structure, supporting lipid bilayer formation and enhancing particle stability. PEG lipids, through their long chains, inhibit non-specific interactions between LNPs and serum proteins, thereby improving biocompatibility and prolonging the in vivo circulating half-life. With these properties, LNPs have become a highly promising gene delivery platform, especially with their successful application in COVID-19 mRNA vaccines, demonstrating their broad therapeutic potential.

[0003] However, traditional lung-targeting peptides (LNPs) exhibit significant hepatic tropism during in vivo delivery, readily accumulating in hepatocytes, which severely limits their application in the treatment of extrahepatic diseases such as lung diseases. To overcome this limitation, current lung-targeting strategies mainly focus on nebulized inhalation, targeted ligand modification, and SORT technology. Nebulized inhalation generates shear forces that easily disrupt the LNP structure, leading to aggregation and imposing stringent requirements on formulation stability. Targeted ligand modification faces challenges such as complex manufacturing processes and high costs, and excessive modification may impair batch-to-batch consistency and reproducibility. While SORT technology can achieve lung enrichment by introducing cationic lipopeptides to regulate surface charge, and offers good targeting and cost control, the large amount of introduced cationic lipopeptides may be accompanied by significant cytotoxicity and inflammatory risks, posing a significant safety concern. Therefore, there is an urgent need to develop a safer, more efficient, and cost-effective lung-targeting LNP delivery system to expand its application in the treatment of respiratory diseases. Summary of the Invention

[0004] Objective of the Invention: The objective of this invention is to provide a lung-targeting LNP based on cationic lipopeptides, its preparation method, and its applications, overcoming the shortcomings of existing technologies. This LNP system can achieve efficient and specific lung targeting through its unique composition without relying on complex ligand modifications or large amounts of cationic lipopeptides. It also possesses good nucleic acid encapsulation efficiency, biocompatibility, and formulation stability, thus providing a safer, more efficient, and easily industrialized delivery tool for gene therapy of lung-related diseases.

[0005] Technical Solution: This invention provides lung-targeting lipid nanoparticles based on cationic lipopeptides. The lipid nanoparticles comprise cationic lipopeptides, ionizable lipids, cholesterol, and PEGylated lipids. The cationic lipopeptides bind to nucleic acids via electrostatic interactions, achieving effective nucleic acid encapsulation. The ionizable lipids are preferably methyl 4-(N,N-dimethylamino)butyrate (dilinyl) methyl ester (DLin-MC3-DMA). The PEGylated lipids are preferably 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol 2000 (DMG-PEG-2000). The cationic lipopeptides consist of a hydrophilic head group and a hydrophobic tail, and the head group characteristics are shown in the following structural formula:

[0006]

[0007] A1 and A2 are independently selected from amino acid residues, specifically arginine, lysine, glutamic acid, histidine, threonine, aspartic acid, and valine.

[0008] When both A1 and A2 are lysine, the head group of the cationic lipopeptide is G2K-OH;

[0009] When both A1 and A2 are arginine, the head group of the cationic lipopeptide is G2R-OH;

[0010] When A1 is lysine and A2 is arginine, the head group of the cationic lipopeptide is M1-OH;

[0011] When A1 is arginine and A2 is lysine, the head group of the cationic lipopeptide is N1-OH;

[0012] This invention provides a method for synthesizing the above-mentioned cationic lipopeptide head group:

[0013] Using H-Lys-OMe·2HCl and Boc-Lys(Boc)-OH as raw materials, amide condensation was carried out in an organic solvent containing a catalyst and an organic base at room temperature and under a nitrogen atmosphere. Finally, Boc-G2K-OMe was obtained by extraction and silica gel column purification.

[0014] Using H-Lys-OMe·2HCl and Boc-Arg(Pbf)-OH as raw materials, amide condensation was carried out in an organic solvent containing a catalyst and an organic base at room temperature and under a nitrogen atmosphere. Finally, Boc,Pbf-G2R-OMe was obtained by extraction and silica gel column chromatography purification.

[0015] Using H-Lys(Fmoc)-OMe·HCl and Boc-Lys(Boc)-OH as raw materials, an intermediate product was prepared in an organic solvent containing a catalyst and an organic base. Then, after Fmoc removal treatment, it was amide condensed with Boc-Arg(Pbf)-OH. Finally, M1-Arg-OMe was obtained by extraction and silica gel column chromatography purification.

[0016] Using Fmoc-Lys-OMe·HCl and Boc-Lys(Boc)-OH as raw materials, an intermediate product was prepared in an organic solvent containing a catalyst and an organic base. Then, after Fmoc removal, it was amide condensed with Boc-Arg(Pbf)-OH. Finally, N1-Arg-OMe was obtained by extraction and silica gel column chromatography purification.

[0017] The preferred condensing agent for the amide condensation of G2R-OMe and Boc-G2K-OMe is a combination of HOBt and HBTU; the preferred condensing agent for the amide condensation of M1-OMe and N1-OMe is a combination of HOBt and EDCI; and the preferred organic base is DIPEA.

[0018] In the preparation of Boc-G2K-OMe, the molar ratio of H-Lys-OMe·2HCl:Boc-Lys(Boc)-OH:HOBt:HBTU:DIPEA is 1:2.2~3:2~5:2~5:6~10; in the preparation of Boc,Pbf-G2R-OMe, the molar ratio of H-Lys-OMe·2HCl:Boc-Arg(Pbf)-OH:HOBt:HBTU:DIPEA is 1:2.2~3:2~5:2~5:6~10. In the preparation of M1-Arg-OMe, the molar ratio of H-Lys(Fmoc)-OMe·HCl:Boc-Lys(Boc)-OH:HOBt:EDCI:DIPEA is 1:1.1~2:2~5:2~5:6~10; in the preparation of N1-Arg-OMe, the molar ratio of Fmoc-Lys-OMe·HCl:Boc-Lys(Boc)·OH:HOBt:EDCI:DIPEA is 1:1.1~2:2~5:2~5:6~10.

[0019] Preferably, in the preparation of Boc-G2K-OMe, the molar ratio of H-Lys-OMe·2HCl:Boc-Lys(Boc)-OH:HOBt:HBTU:DIPEA is 1:2.5:3:3:7.5; in the preparation of Boc,Pbf-G2R-OMe, the molar ratio of H-Lys-OMe·2HCl:Boc-Arg(Pbf)-OH:HOBt:HBTU:DIPEA is 1:3:3:3:7.5; M1 In the preparation of -Arg-OMe, the molar ratio of HLYs(Fmoc)-OMe·HCl:Boc-Lys(Boc)-OH:HOBt:EDCI:DIPEA is 1:1.2:1.5:1.5:10; in the preparation of N1-Arg-OMe, the molar ratio of Fmoc-Lys-OMe·HCl:Boc-Lys(Boc)-OH:HOBt:EDCI:DIPEA is 1:1.2:1.5:1.5:10.

[0020] Boc-G2K-OMe, Boc,Pbf-G2R-OMe, M1-Arg-OMe, and N1-Arg-OMe were added to a methanol solution of NaOH. After the reaction was completed, G2K-OH, G2R-OH, M1-OH, and N-OH were obtained by adjusting the pH and extraction, respectively.

[0021] The concentration of the NaOH in the methanol solution is 0.5–2 mol / L; preferably 1 mol / L.

[0022] This invention provides a method for synthesizing the tail of the above-mentioned cationic lipopeptide, the tail of which has the following structural features as shown in the following formula:

[0023]

[0024] R1 and R2 are organic carbon chains, independently selected from C6-C24 alkyl, C6-C24 alkyl substituted with substituents, C6-C24 alkenyl, C6-C24 alkenyl substituted with substituents, C6-C24 ynyl or substituted C6-C24 ynyl.

[0025] Wherein, when R1 and R2 are C18, representing the hydrophobic tail intermediate C18 of the cationic lipopeptide, the C18 is characterized by being prepared by the following method:

[0026] N-Boc-ethylenediamine was reacted with 1-bromooctadecane under alkaline conditions in an organic solvent to generate an intermediate. The intermediate was then purified by extraction, washing, drying, and silica gel column chromatography. Subsequently, the intermediate was placed in dichloromethane under acidic conditions to remove the Boc protecting group. The deprotected reaction solution was neutralized, extracted, dried, and concentrated to finally obtain the target product C18.

[0027] Preferably, the acid used to remove the Boc protecting group is trifluoroacetic acid.

[0028] This invention provides a method for linking the head and tail of the above-mentioned cationic lipopeptide:

[0029] The tail intermediate C18 and the protected head intermediate were subjected to an amide condensation reaction in an organic solvent in the presence of a condensing agent and an organic base to obtain a protected cationic lipopeptide precursor; the precursor was purified by column chromatography; subsequently, under acidic conditions, the protected cationic lipopeptide precursor was placed in an organic solvent for a deprotection reaction to obtain the target cationic lipopeptide.

[0030] The condensing agent is preferably a combination of HOBt and EDCI, and the organic base is preferably DIPEA; the acidic conditions are provided by trifluoroacetic acid.

[0031] During the head and tail condensation process, the molar ratio of tail intermediate C18: protected head intermediate: HOBt: EDCI: DIPEA is 1:2~3:2~5:2~5:6~10; preferably 1:2.5:3:3:8.

[0032] The cationic lipopeptides are further used to prepare lipid nanoparticles, which can be applied to non-viral nucleic acid carriers.

[0033] The cationic lipopeptide molecule lung-targeting LNP of the present invention can be used for nucleic acid delivery. The nucleic acid drug is characterized by being selected from any one or a combination of several of the following: small interfering RNA (siRNA), messenger RNA (mRNA), microRNA, circular RNA (circRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), guide RNA (gRNA), short hairpin RNA (shRNA), PIWI-interacting RNA (piRNA), repeat-associated small interfering RNA (rasiRNA), heterogeneous nuclear RNA (hnRNA), long non-coding RNA (lncRNA), plasmid DNA, closed circular DNA (ceDNA), mini circle DNA, and antisense oligonucleotides (ASOs).

[0034] This invention provides a method for preparing lung-targeting lipid nanoparticles, the method comprising the following steps:

[0035] Cationic lipopeptides, ionizable lipids, cholesterol, and PEGylated lipids were dissolved in an organic solvent to form a lipid phase; simultaneously, nucleic acids were dissolved in an acidic buffer to form an aqueous phase; the lipid phase and aqueous phase were mixed and thoroughly mixed by vortexing to bring the two phases into contact, and then incubated to self-assemble into lipid nanoparticles encapsulated with nucleic acids; unencapsulated nucleic acids and free lipids were removed by membrane filtration to obtain purified lung-targeting lipid nanoparticles.

[0036] Preferably, the self-assembled product is purified by ultrafiltration, and more preferably by centrifugation at 7000g for 40 minutes.

[0037] Invention Principle: The invention designs and constructs the lung-targeting LNP system based on cationic lipopeptides based on the following core principles:

[0038] The "head-tail" molecular design principle: The cationic lipopeptides (such as G2K-C18, G2R-C18, etc.) designed in this invention adopt a modular design. Their hydrophilic heads are composed of cationic amino acids such as arginine and lysine, which not only efficiently compress and encapsulate negatively charged nucleic acids through electrostatic interactions but also determine the initial chemical properties of the LNP surface. Their hydrophobic tails are composed of long-chain alkyl groups (C18), which can anchor within the lipid bilayer of the LNP, ensuring stable molecular embedding. This design achieves a unity of nucleic acid encapsulation and biological function.

[0039] The principle of in-situ formation of a "targeted protein crown": Unlike traditional ligand-based active targeting strategies, the LNP of this invention, after being injected into the body, has its surface-embedded cationic lipopeptides acting as a "signal source," specifically adsorbing specific biomolecules (such as specific apolipoproteins) in the plasma, forming a unique "protein crown" in situ. This protein crown is not a random layer of non-specifically adsorbed protein, but rather a "targeted navigation system" that can be efficiently recognized by receptors on the surface of lung tissue cells (such as lung endothelial cells and epithelial cells), thereby actively mediating the efficient absorption of LNPs by lung cells, achieving efficient and active enrichment in the lungs.

[0040] The principle of synergistic component and safety balance: The LNP system achieves functional synergy through the precise ratio of four components. The cationic lipopeptide provides targeting while its dosage is optimized at a low level (e.g., 10%) to avoid cytotoxicity caused by excessive cationic charge; ionizable lipids (e.g., DLin-MC3-DMA) ensure protonation in acidic endosomal environments, promoting endosome escape; cholesterol and PEG lipids together maintain the structural stability and long-circulating properties of the LNP. This design achieves efficient lung-targeted delivery while ensuring the safety and stability of the formulation.

[0041] Beneficial effects: Compared with the prior art, the technical solution provided by the present invention has the following significant advantages and positive effects:

[0042] This invention achieves highly efficient, specific, and universal active lung targeting: overcoming the liver-oriented limitation of traditional LNPs. Through a unique mechanism of cationic lipopeptide-induced formation of a specific protein crown, highly efficient enrichment of LNPs in lung tissue is achieved. Animal experiments show that, compared to traditional LNPs that mainly accumulate in the liver, the enrichment signal of the LNPs in the lungs of this invention can be increased by several to tens of times.

[0043] Combining excellent biocompatibility with superior clinical translation potential: This invention fundamentally avoids the cytotoxicity and inflammatory risks associated with the large-scale use of synthetic cationic lipopeptides in SORT technology by introducing structurally well-defined biogenic cationic lipopeptides and optimizing their usage ratio. In vitro cytotoxicity experiments confirmed that the optimal formulation of LNP in this invention achieves highly efficient transfection while maintaining significantly higher cell viability than the high-proportion cationic lipopeptide control group. This enhanced biocompatibility provides a key advantage for its clinical translation.

[0044] The preparation process is simple, cost-controllable, and reproducible: This LNP system does not rely on complex, expensive, and batch-to-batch variability chemical coupling processes. It can be prepared using only a one-step self-assembly process such as conventional microfluidic or ethanol injection methods. This not only simplifies the production steps and significantly reduces production costs, but also significantly improves the batch-to-batch consistency and quality control of the product, making it more suitable for large-scale industrial production.

[0045] High nucleic acid encapsulation efficiency and complete gene delivery: Thanks to the strong positive charge of the cationic lipopeptide head, the LNP of this invention exhibits extremely high encapsulation efficiency for various negatively charged nucleic acids, ensuring the stability of gene drugs during in vivo delivery. From nucleic acid compression and in vivo targeting to endocytosis and endosome escape, this invention provides a complete and efficient gene delivery solution, laying a solid technical foundation for gene therapy and vaccine development for lung diseases. Attached Figure Description

[0046] Figure 1 These are mass spectrometry results of different cationic lipopeptide materials shown in Examples 1-4;

[0047] Figure 2 The particle size distribution diagram of LNPs prepared from different cationic lipopeptides as shown in Example 6;

[0048] Figure 3 The potential characterization diagrams are for LNPs prepared from different cationic lipopeptides as shown in Example 6.

[0049] Figure 4 The combined effect of LNPs prepared from different cationic lipopeptides with nucleic acids as shown in Example 7;

[0050] Figure 5The encapsulation efficiency of LNPs and nucleic acids prepared from different cationic lipopeptides as shown in Example 7;

[0051] Figure 6-7 The in vivo targeted distribution of LNPs prepared from different cationic lipopeptides is shown in Example 8;

[0052] Figure 8 The results of cytotoxicity of the lung-targeting cationic lipopeptide LNP shown in Example 9;

[0053] Figure 9 The results of cellular uptake of lung-targeting cationic lipopeptide LNP as shown in Example 10.

[0054] Figure 10 The results of cell transfection of lung-targeting cationic lipopeptide LNP as shown in Example 11. Detailed Implementation

[0055] The technical solution of the present invention will be further described below with reference to the embodiments. The test materials used in the embodiments can all be purchased through conventional means.

[0056] Example 1

[0057] The cationic dendritic lipopeptide G2K-C18 of the present invention is prepared by the following steps:

[0058]

[0059] Step 1: Accurately weigh H-Lys-OMe·2HCl (2.00 g, 8.58 mmol), Boc-Lys(Boc)-OH (7.43 g, 21.45 mmol), HOBt (3.48 g, 25.74 mmol), and HBTU (9.76 g, 25.74 mmol) into a 250 mL three-necked flask, evacuate, and protect under nitrogen atmosphere. Add DMF (50 mL) to dissolve the reactants. After the reactants are fully dissolved, slowly add DIPEA (11.21 mL, 64.35 mmol) and react for 24 hours. After the reaction is complete, remove the solvent using a rotary evaporator, dissolve the residue with ethyl acetate, and then extract three times successively with saturated sodium bicarbonate solution, 0.1% dilute HCl solution, and saturated NaCl solution. Dry the organic phase with anhydrous sodium sulfate to remove water, filter, and vacuum rotary evaporate to obtain crude Boc-G2K-OMe.

[0060] Step 2: The crude Boc-G2K-OMe product from Step 1 was purified by column chromatography with DCM:MeOH = 40:1 and V:V elution. The solvent was removed by rotary evaporation to obtain the Boc-G2K-OMe product. The obtained product was dried in a vacuum drying oven for 12 hours until completely dry.

[0061] Step 3: Accurately weigh 2.00 g of Boc-G2K-OMe into a 50 mL round-bottom flask, evacuate, and protect under nitrogen atmosphere. Add a methanol solution containing 1 M NaOH under ice bath conditions. After reacting at room temperature for 6 h, adjust the pH to 2-3 with 0.1 M HCl, remove the solvent by rotary evaporation, dissolve in ethyl acetate, extract with saturated NaCl solution, dry the organic phase with anhydrous sodium sulfate to remove water, filter, and then rotary evaporate under vacuum to obtain crude Boc-G2K-OH.

[0062] Step 4: Under nitrogen protection, N-Boc-Z diamine (2.42 g, 15.09 mmol), 1-bromooctadecane (12.58 g, 37.73 mmol), potassium carbonate (5.21 g, 37.73 mmol), and potassium iodide (1.75 g, 15.09 mmol) were dissolved in N,N-dimethylformamide. The reaction mixture was stirred at 80 °C for 24 hours. After the reaction was complete, the solvent was evaporated under reduced pressure, and the residue was dissolved in ethyl acetate. The organic phase was washed successively with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate for 2 hours, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was purified by normal-phase silica gel column chromatography with dichloromethane / methanol (90:1, V / V) as the eluent to obtain compound 1. Under nitrogen protection and an ice bath, compound 1 (2.00 g, 3.01 mmol) was dissolved in dichloromethane, and trifluoroacetic acid (65.29 mmol) was slowly added. The reaction mixture was brought to room temperature and stirred for another 2 hours. After the reaction was complete, the solvent was removed under reduced pressure, and the residue was dissolved in ethyl acetate. The organic phase was washed successively with saturated sodium bicarbonate aqueous solution, dried over anhydrous sodium sulfate for 2 hours, filtered, and the filtrate was concentrated under reduced pressure to give the final product C18.

[0063] Step 5: Accurately weigh C18 (1.25 g, 2.65 mmol), Boc-G2K-OH (0.85 g, 1.06 mmol), HOBt (0.36 g, 2.66 mmol), and EDCI (0.509 g, 2.66 mmol) into a 250 mL three-necked flask, evacuate, and protect under nitrogen atmosphere. Add DMF (50 mL) to dissolve the reactants. After the reactants are fully dissolved, slowly add DIPEA (1.22 mL, 7.09 mmol) and react for 12 hours. After the reaction is complete, remove the solvent using a rotary evaporator, dissolve the residue with ethyl acetate, and then extract three times successively with saturated sodium bicarbonate solution, 0.1% dilute HCl solution, and saturated NaCl solution. Dry the organic phase with anhydrous sodium sulfate to remove water, filter, and vacuum rotary evaporate to obtain crude Boc-G2K-C18.

[0064] Step 6: The crude Boc-G2K-C18 product from Step 5 was purified by column chromatography with DCM:MeOH = 30:1 and V:V elution. The solvent was removed by rotary evaporation to obtain the Boc-G2K-C18 product. The obtained product was dried in a vacuum drying oven for 12 hours until completely dry.

[0065] Step 7: Accurately weigh 200 mg of Boc-G2K-C18 into a 50 mL round-bottom flask, evacuate, and protect under nitrogen atmosphere. Add 2 mL each of anhydrous dichloromethane and TFA in an ice bath. After reacting in an ice bath for 2 hours, wash three times with cold diethyl ether, remove the supernatant, and dry the precipitate in a vacuum drying oven to obtain G2K-C18.

[0066] 1H NMR (300MHz, DMSO) δ 8.75-8.13 (m, 7H), 7.87 (s, 4H), 4.27-3.42 (m, 5H), 3.08 (s, 8H), 2.75 (s, 4H), 1.24 (s, 82H), 0.91-0.81 (m, 6H).

[0067] Example 2

[0068] The cationic dendritic lipopeptide G2R-C18 of the present invention is prepared by the following steps:

[0069]

[0070] Step 1: Accurately weigh Boc-Arg(Pbf)-OH (13.56 g, 25.74 mmol), H-Lys-OMe-2HCl (2.00 g, 8.58 mmol), HOBt (3.48 g, 25.74 mmol), and HBTU (9.76 g, 25.74 mmol) into a 250 mL three-necked flask, evacuate, and protect under nitrogen atmosphere. Add DMF (120 mL) to dissolve the reactants. After the reactants are fully dissolved, slowly add DIPEA (11.95 mL, 68.64 mmol) and react for 24 hours. After the reaction is complete, remove the solvent using a rotary evaporator, dissolve the residue with ethyl acetate, and then extract three times successively with saturated sodium bicarbonate solution, 0.1% dilute HCl solution, and saturated NaCl solution. Dry the organic phase with anhydrous sodium sulfate to remove water, filter, and then vacuum evaporate to obtain crude Boc,Pbf-G2R-OMe.

[0071] Step 2: The crude Boc,Pbf-G2R-OMe product from Step 1 was purified by column chromatography. The dimethyl methacrylate (DCM):MeOH = 30:1, V:V elution was used. After removing the solvent by rotary evaporation, the Boc,Pbf-G2R-OMe product was obtained. The obtained product was dried in a vacuum drying oven for 12 hours until completely dry.

[0072] Step 3: Accurately weigh 2.00 g of Boc,Pbf-G2R-OMe into a 50 mL round-bottom flask, evacuate, and protect under nitrogen atmosphere. Add a methanol solution containing 1 M NaOH under ice bath conditions. After reacting at room temperature for 6 h, adjust the pH to 2-3 with 0.1 M HCl, remove the solvent by rotary evaporation, dissolve in ethyl acetate, extract with saturated NaCl solution, dry the organic phase with anhydrous sodium sulfate to remove water, filter, and then rotary evaporate under vacuum to obtain crude Boc,Pbf-G2R-OH.

[0073] Step 4: Under nitrogen protection, N-Boc-ethylenediamine (2.42 g, 15.09 mmol), 1-bromooctadecane (12.58 g, 37.73 mmol), potassium carbonate (5.21 g, 37.73 mmol), and potassium iodide (1.75 g, 15.09 mmol) were dissolved in N,N-dimethylformamide. The reaction mixture was stirred at 80 °C for 24 hours. After the reaction was complete, the solvent was evaporated under reduced pressure, and the residue was dissolved in ethyl acetate. The organic phase was washed successively with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate for 2 hours, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was purified by normal-phase silica gel column chromatography with dichloromethane / methanol (90:1, V / V) as the eluent to obtain compound 1. Under nitrogen protection and an ice bath, compound 1 (2.00 g, 3.01 mmol) was dissolved in dichloromethane, and trifluoroacetic acid (65.29 mmol) was slowly added. The reaction mixture was brought to room temperature and stirred for another 2 hours. After the reaction was complete, the solvent was removed under reduced pressure, and the residue was dissolved in ethyl acetate. The organic phase was washed successively with saturated sodium bicarbonate aqueous solution, dried over anhydrous sodium sulfate for 2 hours, filtered, and the filtrate was concentrated under reduced pressure to give the final product C18.

[0074] Step 5: Accurately weigh C18 (1.25 g, 2.65 mmol), Boc,Pbf-G2R-OH (1.24 g, 1.06 mmol), HOBt (0.36 g, 2.66 mmol), and EDCI (0.51 g, 2.66 mmol) into a 250 mL three-necked flask, evacuate, and protect under nitrogen atmosphere. Add DMF (50 mL) to dissolve the reactants. After the reactants are fully dissolved, slowly add DIPEA (1.22 mL, 7.09 mmol) and react for 12 hours. After the reaction is complete, remove the solvent using a rotary evaporator, dissolve the residue with ethyl acetate, and then extract three times successively with saturated sodium bicarbonate solution, 0.1% dilute HCl solution, and saturated NaCl solution. Dry the organic phase with anhydrous sodium sulfate to remove water, filter, and then vacuum evaporate to obtain crude Boc,Pbf-G2R-C18.

[0075] Step 6: The crude Boc,Pbf-G2R-C18 product from Step 5 was purified by column chromatography. The dimethyl methacrylate (DCM):MeOH = 25:1, V:V elution was used. After removing the solvent by rotary evaporation, the Boc,Pbf-G2R-C18 product was obtained. The obtained product was dried in a vacuum drying oven for 12 hours until completely dry.

[0076] Step 7: Accurately weigh 200 mg of Boc,Pbf-G2R-C18 into a 50 mL round-bottom flask, evacuate, and protect under nitrogen atmosphere. Add 2 mL each of anhydrous dichloromethane and TFA in an ice bath. After reacting in an ice bath for 24 h, wash three times with cold diethyl ether, remove the supernatant, and dry the precipitate in a vacuum drying oven to obtain G2R-C18.

[0077] 1H NMR (300MHz, DMSO) δ8.59-7.23 (m, 15H), 4.26-3.41 (m, 7H), 3.11 (s, 10H), 1.23 (s, 78H), 0.90-0.81 (m, 6H).

[0078] Example 3

[0079] The cationic dendritic lipopeptide M1-C18 of the present invention is prepared by the following steps:

[0080]

[0081] Step 1: Accurately weigh H-Lys(Fmoc)-OMe·HCl (6.00 g, 14.32 mmol), Boc-Lys(Boc)-OH (5.95 g, 17.19 mmol), HOBt (2.90 g, 21.48 mmol), and EDCI (4.10 g, 21.48 mmol) into a 250 mL three-necked flask, evacuate, and protect under nitrogen atmosphere. Add DMF (150 mL) to dissolve the reactants. After the reactants are fully dissolved, slowly add DIPEA (24.86 mL, 140.32 mmol) and react for 24 hours. After the reaction is complete, remove the solvent using a rotary evaporator, dissolve the residue with ethyl acetate, and then extract three times successively with saturated sodium bicarbonate solution, 0.1% dilute HCl solution, and saturated NaCl solution. Dry the organic phase with anhydrous sodium sulfate to remove water, filter, and vacuum rotary evaporate to obtain crude Fmoc-M1-OMe.

[0082] Step 2: The crude Fmoc-M1-OMe product from Step 1 was purified by column chromatography (DCM:MeOH = 70:1, V:V) elution. After removing the solvent by rotary evaporation, the Fmoc-M1-OMe product was obtained. The obtained product was dried in a vacuum drying oven for 12 hours until completely dry.

[0083] Step 3: Accurately weigh 2.00 g of Fmoc-M1-OMe into a 50 mL round-bottom flask, evacuate, and protect under nitrogen atmosphere. Add piperidine / DMF mixed solution under ice bath conditions. After reacting at room temperature for 30 min, repeatedly remove the solvent and piperidine by rotary evaporation to obtain crude M1-OMe. Purify the crude product by column chromatography with DCM:MeOH = 70:1, V:V elution. After removing the solvent by rotary evaporation, obtain the M1-OMe product. Dry the obtained product in a vacuum drying oven for 12 h until completely dry.

[0084] Step 4: Accurately weigh M1-OMe (3.74 g, 7.66 mmol), Boc-Arg(Pbf)-OH (4.84 g, 9.19 mmol), HOBt (1.55 g, 11.49 mmol), and HBTU (4.36 g, 11.49 mmol) into a 250 mL three-necked flask, evacuate, and protect under nitrogen atmosphere. Add DMF (140 mL) to dissolve the reactants. After the reactants are fully dissolved, slowly add DIPEA (10.66 mL, 61.28 mmol) and react for 12 hours. After the reaction is complete, remove the solvent using a rotary evaporator, dissolve the residue with ethyl acetate, and then extract three times successively with saturated sodium bicarbonate solution, 0.1% dilute HCl solution, and saturated NaCl solution. Dry the organic phase with anhydrous sodium sulfate to remove water, filter, and then vacuum evaporate to obtain crude M1-Arg-OMe.

[0085] Step 5: The crude M1-Arg-OMe product from Step 4 was purified by column chromatography with DCM:MeOH = 30:1 and V:V elution. The solvent was removed by rotary evaporation to obtain the M1-Arg-OMe product. The obtained product was dried in a vacuum drying oven for 12 hours until completely dry.

[0086] Step 6: Accurately weigh 2.00 g of M1-Arg-OMe into a 50 mL round-bottom flask, evacuate, and protect under nitrogen atmosphere. Add a methanol solution containing 1 M NaOH under ice bath conditions. After reacting at room temperature for 6 h, adjust the pH to 2-3 with 0.1 M HCl, remove the solvent by rotary evaporation, dissolve in ethyl acetate, extract with saturated NaCl solution, dry the organic phase with anhydrous sodium sulfate to remove water, filter, and then rotary evaporate under vacuum to obtain crude M1-Arg-OH.

[0087] Step 7: Under nitrogen protection, N-Boc-ethylenediamine (2.42 g, 15.09 mmol), 1-bromooctadecane (12.58 g, 37.73 mmol), potassium carbonate (5.21 g, 37.73 mmol), and potassium iodide (1.75 g, 15.09 mmol) were dissolved in N,N-dimethylformamide. The reaction mixture was stirred at 80 °C for 24 hours. After the reaction was complete, the solvent was evaporated under reduced pressure, and the residue was dissolved in ethyl acetate. The organic phase was washed successively with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate for 2 hours, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was purified by normal-phase silica gel column chromatography with dichloromethane / methanol (90:1, V / V) as the eluent to obtain compound 1. Under nitrogen protection and an ice bath, compound 1 (2.00 g, 3.01 mmol) was dissolved in dichloromethane, and trifluoroacetic acid (65.29 mmol) was slowly added. The reaction mixture was brought to room temperature and stirred for another 2 hours. After the reaction was complete, the solvent was removed under reduced pressure, and the residue was dissolved in ethyl acetate. The organic phase was washed successively with saturated sodium bicarbonate aqueous solution, dried over anhydrous sodium sulfate for 2 hours, filtered, and the filtrate was concentrated under reduced pressure to give the final product C18.

[0088] Step 8: Accurately weigh C18 (1.25 g, 2.65 mmol), M1-Arg-OH (1.05 g, 1.06 mmol), HOBT (0.36 g, 2.66 mmol), and EDCI (0.51 g, 2.66 mmol) into a 250 mL three-necked flask, evacuate, and protect under nitrogen atmosphere. Add DMF (50 mL) to dissolve the reactants. After the reactants are fully dissolved, slowly add DIPEA (1.23 mL, 7.08 mmol) and react for 12 hours. After the reaction is complete, remove the solvent using a rotary evaporator, dissolve the residue with ethyl acetate, and then extract three times successively with saturated sodium bicarbonate solution, 0.1% dilute HCl solution, and saturated NaCl solution. Dry the organic phase with anhydrous sodium sulfate to remove water, filter, and then vacuum evaporate to obtain crude Boc,Pbf-M1-C18.

[0089] Step 9: The crude Boc,Pbf-M1-C18 product from Step 8 was purified by column chromatography with DCM:MeOH = 25:1 and V:V elution. After removing the solvent by rotary evaporation, the Boc,Pbf-M1-C18 product was obtained. The obtained product was dried in a vacuum drying oven for 12 hours until completely dry.

[0090] Step 10: Accurately weigh 200 mg of Boc, Pbf-M1-C18 into a 50 mL round-bottom flask, evacuate, and protect under nitrogen atmosphere. Add 2 mL each of anhydrous dichloromethane and TFA in an ice bath. After reacting in an ice bath for 24 h, wash three times with cold diethyl ether, remove the supernatant, and dry the precipitate in a vacuum drying oven to obtain M1-C18.

[0091] 1H NMR (300MHz, DMSO) δ 8.76-7.84 (m, 11H), 7.32 (s, 2H), 4.27-3.37 (m, 5H), 3.07 (s, 10H), 2.74 (s, 2H), 1.23 (s, 80H), 0.85 (t, J = 6.3Hz, 6H).

[0092] Example 4

[0093] The cationic dendritic lipopeptide N1-C18 of the present invention is prepared by the following steps:

[0094]

[0095] Step 1: Accurately weigh Fmoc-Lys-OMe·HCl (6.00 g, 14.32 mmol), Boc-Lys(Boc)-OH (5.95 g, 17.19 mmol), HOBt (2.90 g, 21.48 mmol), and EDCI (4.10 g, 21.48 mmol) into a 250 mL three-necked flask, evacuate, and protect under nitrogen atmosphere. Add DMF (60 mL) to dissolve the reactants. After the reactants are fully dissolved, slowly add DIPEA (24.86 mL, 140.32 mmol) and react for 24 hours. After the reaction is complete, remove the solvent using a rotary evaporator, dissolve the residue with ethyl acetate, and then extract three times successively with saturated sodium bicarbonate solution, 0.1% dilute HCl solution, and saturated NaCl solution. Dry the organic phase with anhydrous sodium sulfate to remove water, filter, and vacuum rotary evaporate to obtain crude Fmoc-N1-OMe.

[0096] Step 2: The crude Fmoc-N1-OMe product from Step 1 was purified by column chromatography with DCM:MeOH = 60:1 and V:V elution. The solvent was removed by rotary evaporation to obtain the Fmoc-N1-OMe product. The obtained product was dried in a vacuum drying oven for 12 hours until completely dry.

[0097] Step 3: Accurately weigh 3.00 g of Fmoc-N1-OMe into a 50 mL round-bottom flask, evacuate, and protect under nitrogen atmosphere. Add piperidine / DMF mixed solution under ice bath conditions. After reacting at room temperature for 30 min, repeatedly remove the solvent and piperidine by rotary evaporation to obtain crude N1-OMe. Purify the crude product by column chromatography with DCM:MeOH = 70:1, V:V elution. After removing the solvent by rotary evaporation, obtain the N1-OMe product. Dry the obtained product in a vacuum drying oven for 12 h until completely dry.

[0098] Step 4: Accurately weigh N1-OMe (2.96 g, 6.06 mmol), Boc-Arg(Pbf)-OH (3.83 g, 7.27 mmol), HOBt (1.23 g, 9.09 mmol), and HBTU (3.45 g, 9.09 mmol) into a 250 mL three-necked flask, evacuate, and protect under nitrogen atmosphere. Add DMF (140 mL) to dissolve the reactants. After the reactants are fully dissolved, slowly add DIPEA (8.44 mL, 48.46 mmol) and react for 12 hours. After the reaction is complete, remove the solvent using a rotary evaporator, dissolve the residue with ethyl acetate, and then extract three times successively with saturated sodium bicarbonate solution, 0.1% dilute HCl solution, and saturated NaCl solution. Dry the organic phase with anhydrous sodium sulfate to remove water, filter, and vacuum evaporate to obtain crude N1-Arg-OMe.

[0099] Step 5: The crude N1-Arg-OMe product from Step 4 was purified by column chromatography with a DCM:MeOH ratio of 35:1 and a V:V washing ratio. The solvent was removed by rotary evaporation to obtain the N1-Arg-OMe product. The obtained product was dried in a vacuum drying oven for 12 hours until completely dry.

[0100] Step 6: Accurately weigh 1.50 g of N1-Arg-OMe into a 50 mL round-bottom flask, evacuate, and protect under nitrogen atmosphere. Add a methanol solution containing 1 M NaOH under ice bath conditions. After reacting at room temperature for 6 h, adjust the pH to 2-3 with 0.1 M HCl, remove the solvent by rotary evaporation, dissolve in ethyl acetate, extract with saturated NaCl solution, dry the organic phase with anhydrous sodium sulfate to remove water, filter, and then evaporate under vacuum to obtain crude N1-Arg-OH.

[0101] Step 7: Under nitrogen protection, N-Boc-ethylenediamine (2.42 g, 15.09 mmol), 1-bromooctadecane (12.58 g, 37.73 mmol), potassium carbonate (5.21 g, 37.73 mmol), and potassium iodide (1.75 g, 15.09 mmol) were dissolved in N,N-dimethylformamide. The reaction mixture was stirred at 80 °C for 24 hours. After the reaction was complete, the solvent was evaporated under reduced pressure, and the residue was dissolved in ethyl acetate. The organic phase was washed successively with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate for 2 hours, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was purified by normal-phase silica gel column chromatography with dichloromethane / methanol (90:1, V / V) as the eluent to obtain compound 1. Under nitrogen protection and an ice bath, compound 1 (2.00 g, 3.01 mmol) was dissolved in dichloromethane, and trifluoroacetic acid (65.29 mmol) was slowly added. The reaction mixture was brought to room temperature and stirred for another 2 hours. After the reaction was complete, the solvent was removed under reduced pressure, and the residue was dissolved in ethyl acetate. The organic phase was washed successively with saturated sodium bicarbonate aqueous solution, dried over anhydrous sodium sulfate for 2 hours, filtered, and the filtrate was concentrated under reduced pressure to give the final product C18.

[0102] Step 8: Accurately weigh C18 (1.25 g, 2.65 mmol), Pbf-N1-OH (1.05 g, 1.06 mmol), HOBT (0.36 g, 2.66 mmol), and EDCI (0.51 g, 2.66 mmol) into a 250 mL three-necked flask, evacuate, and protect under nitrogen atmosphere. Add DMF (50 mL) to dissolve the reactants. After the reactants are fully dissolved, slowly add DIPEA (1.23 mL, 7.08 mmol) and react for 12 hours. After the reaction is complete, remove the solvent using a rotary evaporator, dissolve the residue with ethyl acetate, and then extract three times successively with saturated sodium bicarbonate solution, 0.1% dilute HCl solution, and saturated NaCl solution. Dry the organic phase with anhydrous sodium sulfate to remove water, filter, and vacuum rotary evaporate to obtain crude Boc,Pbf-N1-C18.

[0103] Step 9: The crude Boc,Pbf-N1-C18 product from Step 8 was purified by column chromatography. The dimethyl methacrylate (DCM):MeOH = 25:1, V:V elution was used. After removing the solvent by rotary evaporation, the Boc,Pbf-N1-C18 product was obtained. The obtained product was dried in a vacuum drying oven for 12 hours until completely dry.

[0104] Step 10: Accurately weigh 200 mg of Boc,Pbf-N1-C18 into a 50 mL round-bottom flask, evacuate, and protect under nitrogen atmosphere. Add 2 mL each of anhydrous dichloromethane and TFA in an ice bath. After reacting in an ice bath for 24 h, wash three times with cold diethyl ether, remove the supernatant, and dry the precipitate in a vacuum drying oven to obtain N1-C18.

[0105] 1H NMR (300MHz, DMSO) δ8.71-7.19 (m, 13H), 4.24-3.40 (m, 5H), 3.14-2.97 (m, 10H), 2.73 (d, J=8.2Hz, 2H), 1.23 (s, 80H), 0.87-0.82 (m, 6H).

[0106] Example 5

[0107] The cationic lipopeptide LNP of the present invention is prepared by the following steps:

[0108] The cationic dendritic lipopeptides prepared in Examples 1-4 were mixed with DLin-MC3-DMA, cholesterol, and DMG-PEG2000 in the prescribed ratio to prepare a lipid phase. The prescribed amount of nucleic acid solution was added to citrate buffer to prepare an aqueous phase. The lipid phase was then quickly added to the aqueous phase, rapidly pipetted 30 times, vortexed for 1 minute, and finally incubated at room temperature for 20 minutes.

[0109] After settling, the LNP was ultrafiltered using an ultrafiltration tube to remove nucleic acids and lipids that did not form LNP. The ultrafiltration tube was placed in a centrifuge and ultrafiltered at 7000xg and 4°C for 40 minutes. The liquid in the inner liner tube was then aspirated to obtain cationic lipopeptide LNP.

[0110] Table 1. LNP prescription ratios for each group

[0111]

[0112] Example 6

[0113] Particle size potential determination of cationic lipopeptide LNP:

[0114] The cationic lipopeptide (LNP) was prepared according to the molar amount specified in the formulation and subjected to ultrafiltration under the conditions of Example 5. The treated LNP was transferred to centrifuge tubes and diluted to 1 mL with HEPES buffer. Then, it was placed in a Malvern plastic sample cell and a potential sample cell, and then placed in the pores of a laser particle size analyzer. The particle size and potential of the nanomedicine were measured at 25°C. Each measurement was performed in triplicate.

[0115] according to Figure 2 and Figure 3The results showed that the particle size and potential met the design expectations. The particle size distribution was concentrated, and the surface charge of LNPs increased with the increase of the molar ratio of cationic lipopeptides. Therefore, this system can effectively adsorb specific biomolecules in plasma, forming a protein corona, and promote the endocytosis of LNPs by lung cells through the corona structure, ultimately achieving excellent lung targeting and enrichment effects.

[0116] Example 7

[0117] Encapsulation effect and encapsulation efficiency determination of cationic lipopeptide LNP:

[0118] The nucleic acid complexation capacity of lipid nanoparticles (LNPs) was assessed using agarose gel electrophoresis. Ultrafiltration was performed according to the conditions in Example 5. LNPs were mixed with loading buffer, and 0.50 g of agarose powder was accurately weighed and placed in 50 mL of UP water. The mixture was boiled and allowed to stand. When the agarose solution cooled to approximately 50°C, 10 μL of GelRed staining solution was added. After thorough mixing, the mixture was poured into a mold to remove air bubbles and allowed to stand for 30 min until solidified. The solidified solution was then transferred to an electrophoresis tank containing TAE buffer. Electrophoresis was performed at 180 V for 30 minutes. mRNA was used as a negative control. After electrophoresis, gel images were acquired using a gel imaging system.

[0119] Using Strand Brite TM RNA quantification reagents were used to determine the encapsulation efficiency (EE) of mRNA. Lipid nanoparticles (LNPs) were incubated for 3 minutes in buffer with or without 2% (v / v) Triton X-100. Then, LNP samples and RNA standards were mixed with StrandBrite. TM Green working solution was mixed. Fluorescence intensity was measured using a multimode microplate reader (excitation / emission wavelength = 490 / 545 nm). Samples treated with Triton X-100 represent the total mRNA content, while untreated samples correspond to the free mRNA content. EE was calculated using the following formula:

[0120]

[0121] like Figure 4-5 As shown, agarose gel electrophoresis results indicate that cationic lipopeptides (LNPs) can effectively encapsulate nucleic acids; encapsulation efficiency measurements show that all cationic lipopeptides (LNPs) exhibit high encapsulation efficiency, with encapsulation efficiencies all >90%.

[0122] Example 8

[0123] Organ-targeted screening of cationic lipopeptide LNP:

[0124] To verify the lung-targeting effect of the LNP described in this invention, mRNA was selected as the model nucleic acid and its in vivo distribution was studied.

[0125] When delivering luciferase messenger RNA (Luc mRNA) in vivo, LNPs encapsulating Luc mRNA are delivered at a dose of 0.1 mg / kg. -1 The drug was administered intravenously to C57BL / 6 mice at a dose specified in the drug. Six hours after injection, the mice were anesthetized with 2% isoflurane. Subsequently, the drugs were administered at a dose of 150 mg / kg. -1 The dosage is 30 mg / mL of D-fluorescein potassium salt administered intraperitoneally. -1 Then, the major organs were imaged using the IVIS Lumina in vivo imaging system (Tianneng ABLX6), and the bioluminescence signal intensity was semi-quantitatively analyzed based on the acquired images.

[0126] like Figure 6 and Figure 7 As shown, firstly, by fixing the molar ratio of cationic lipopeptides, the organ-targeting properties of different cationic lipopeptides were screened. The results showed that G2K-C18 exhibited the best lung-targeting ability. Under the condition of fixing the cationic lipopeptide G2K-C18, the molar ratio of the cationic lipopeptides was adjusted to screen for the formulation that could achieve the best lung-targeting effect. The results showed that the lung-targeting ability gradually increased with the increase of the cationic lipopeptide molar ratio. When the molar ratio reached 30%, the lung-targeting ability was significantly better than the control group. At a molar ratio of 20%, both good targeting effect and high safety were observed.

[0127] Example 9

[0128] In vitro safety evaluation of cationic lipopeptide LNP:

[0129] A549 cells were distributed at a rate of 8 × 10⁸ cells per well. 3 Cells were seeded at a density of 100 μL in 96-well plates and cultured at 37°C and 5% CO2 for 24 hours. LNPs were prepared and diluted to the desired concentration in DMEM. The original medium was then replaced with medium containing different concentrations of LNPs. After 6 hours of incubation, the medium was replaced with fresh DMEM containing 10% fetal bovine serum, and the cells were cultured for another 18 hours. After incubation, the cells were washed with PBS. Subsequently, 100 μL of MTT solution (1 mg / mL) was added to each well. -1 The culture plate was then incubated in the dark for 4 hours. The MTT solution was then carefully removed. After washing with PBS, 150 μL LDMSO was added to each well to dissolve the formazan crystals. Finally, the absorbance (OD) at 490 nm was measured using a microplate reader (Molecular Devices, SpectraMax M2e).

[0130] like Figure 9 As shown, the final lung-targeting cationic lipopeptide (LNP) maintained a high cell viability (>80%) even at a dose of 100 ng. In contrast, the cell viability decreased to 80% at a dose of 75 ng. This result indicates that the lung-targeting cationic lipopeptide (LNP) exhibits good biocompatibility while ensuring its targeting efficacy.

[0131] Example 10

[0132] Cellular uptake capacity assessment:

[0133] A549 cells were spaced at 5 × 10⁶ cells per well. 4 Cells were seeded at a density of [number] cells / well in 24-well plates and incubated at 37°C with 5% CO2 for 24 hours to allow complete cell adhesion. Subsequently, Cy5-labeled samples of DOTAP and G2K-20% were prepared. The original culture medium was removed from the wells, and the cells were gently washed twice with PBS. Then, 500 μL of fresh serum-free culture medium containing an equal volume of Cy5-labeled drug was added to each well, and the cells were incubated at 37°C for 2 hours and 6 hours, respectively.

[0134] After the predetermined incubation time, the drug-containing culture medium was completely removed, and the cells were washed three times thoroughly with pre-chilled PBS buffer to remove any untaken nanoparticles. Subsequently, trypsin was added to each well for digestion, and the cells were collected and centrifuged at 1000 rpm for 5 minutes, discarding the supernatant. Finally, the cells were resuspended in 200 μL of pre-chilled PBS and immediately analyzed by flow cytometry. The cell uptake efficiency of different formulations at different time points was quantitatively evaluated by analyzing Cy5 fluorescence intensity.

[0135] like Figure 9 As shown, compared with the control group, the lung-targeting cationic lipopeptide (LNP) not only exhibited superior targeting efficacy but also maintained significantly higher cellular uptake capacity. This indicates that the LNP system can be effectively taken up by lung cells while targeting them, thus ensuring its high efficiency and bioavailability in targeted therapy. These results further validate the dual advantages of lung-targeting LNP in improving drug delivery efficiency and ensuring uptake by targeted cells.

[0136] Example 11

[0137] Evaluation of the transfection capability of dendritic lipopeptide nanomedicines:

[0138] A549-Luc cells were planted at a density of 8 × 10⁶ cells per well. 3Cells were seeded at a density of 100 g / well in 96-well plates and cultured at 37°C under a 5% CO2 atmosphere for 24 h. shLuc lipid nanoparticles (LNPs) were prepared and diluted to the desired concentration in DMEM medium. 100 ng of shLuc was added to each well. The original culture medium was removed and replaced with diluted medium containing LNPs. After incubating the cells with LNPs at 37°C for 6 h, the medium was replaced with fresh DMEM medium containing 10% fetal bovine serum (FBS), and cultured for another 18 h. After incubation, cell lysates were collected. Luciferase activity was measured using a luciferase reporter gene assay kit (Beyotime, China), and the relative protein concentration of the lysates was determined using a BCA protein assay kit (Beyotime, China). Luciferase activity is expressed as relative light units (RLU). Relative expression rate (%) was calculated using the following formula:

[0139]

[0140] like Figure 10 As shown, compared with the control group, this lung-targeting cationic lipopeptide (LNP) not only maintained good targeting ability but also exhibited excellent cell transfection capability. This indicates that the LNP system can efficiently deliver nucleic acid or genetic materials into target cells and achieve effective gene expression. Such transfection effect provides a solid foundation for the implementation of gene therapy, opens up new possibilities for the treatment of lung-related diseases, and further demonstrates its potential in targeted therapy.

Claims

1. A lung-targeting lipid nanoparticle (LNP) based on cationic lipopeptides, characterized in that, The composition includes ionizable lipids, cationic lipopeptides or auxiliary lipids, cholesterol, and PEGylated lipids, which are prepared by nanoprecipitation; wherein the cationic lipopeptides serve as functional components and can replace auxiliary lipids.

2. The cationic lipopeptide lung-targeting lipid nanoparticles according to claim 1, characterized in that, The ionizable lipid is any one of the following: 4-(N,N-dimethylamino)butyrate (dilinyl) methyl ester (DLin-MC3-DMA), ((4-hydroxybutyl)azadiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), 3-[4,4-bis(octyloxy)-1-oxobutoxy]-2-[[[3-(diethylamino)propoxy]carbonyl]oxy]methyl]propyl(9Z,12Z)-9,12-octadecadienoic acid (LP-01), 1,1′-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azadiyl)bis(dodecane-2-ol) (C12-200).

3. The cationic lipopeptide lung-targeting lipid nanoparticles according to claim 1, characterized in that, The cationic lipopeptides are G2K-C18, G2R-C18, M1-C18, and N1-C18, respectively, and their structural features are shown in formula a: R1 and R2 are organic carbon chains, independently selected from C6-C24 alkyl, C6-C24 alkyl substituted with substituents, C6-C24 alkenyl, C6-C24 alkenyl substituted with substituents, C6-C24 ynyl or substituted C6-C24 ynyl. A1 and A2 are independently selected from amino acid residues, specifically arginine, lysine, glutamic acid, histidine, threonine, aspartic acid, and valine. When R1 and R2 are C18 and A1 and A2 are both lysine, the cationic lipopeptide is named G2K C18 and its structure is shown in formula a1. When R1 and R2 are C18 and A1 and A2 are both arginine, the cationic lipopeptide is named G2R-C18 and its structure is shown in formula a2. When R1 and R2 are C18, A1 is lysine, and A2 is arginine, the cationic lipopeptide is named M1-C18, and its structure is shown in formula a3. When R1 and R2 are C18, A1 is arginine, and A2 is lysine, the cationic lipopeptide is named N1.C18, and its structure is shown in formula a4. G2K-C18: G2R-C18: M1-C18: N1-C18:

4. The four cationic lipopeptides according to claim 3, characterized in that, The cationic lipopeptide comprises a hydrophilic dendritic amino acid head group and a hydrophobic alkane chain fatty tail; wherein the alkane chain tail is composed of a nonpolar hydrocarbon chain and has a hydrophobic structure, which is used to form an amphiphilic lipopeptide molecule together with the hydrophilic head group.

5. The four cationic lipopeptides according to claim 3, characterized in that, The method for preparing the amino acid head group of the cationic lipopeptide is as follows: When synthesizing G2K-C18 and G2R-C18, the target amino acid head group is obtained by one-step amide condensation and alkaline hydrolysis reaction of H-Lys-OMe·2HCl with the corresponding Boc protected amino acid; when synthesizing M1-C18 and N1-C18, the head sequence is constructed by starting with the corresponding Fmoc protected amino acid and proceeding through amide condensation, deprotection, and re-condensation steps, and finally obtained by alkaline hydrolysis reaction. Furthermore, the present invention provides a method for preparing the above-mentioned amino acid head group: Using H-Lys-OMe·2HCl and Boc-Lys(Boc)-OH as raw materials, an amide condensation reaction was carried out in an inert atmosphere in the presence of a condensing agent and an organic base. The reaction product was purified by extraction and silica gel column chromatography to obtain Boc-G2K-OMe. Using H-Lys-OMe·2HCl and Boc-Arg(Pbf)-OH as raw materials, an amide condensation reaction was carried out in an inert atmosphere in the presence of a condensing agent and an organic base. The reaction product was purified by extraction and silica gel column chromatography to obtain Boc,Pbf-G2R-OMe. Using H-Lys(Fmoc)-OMe·HCl and Boc-Lys(Boc)-OH as raw materials, a first-step amide condensation was carried out in the presence of a condensing agent and an organic base to obtain an intermediate product. Subsequently, the intermediate product was deprotected from Fmoc and then subjected to a second-step amide condensation with Boc-Arg(Pbf)-OH in the presence of a condensing agent and an organic base. The reaction product was purified by extraction and silica gel column chromatography to obtain M1-Arg-OMe. Using Fmoc-Lys-OMe·HCl and Boc-Lys(Boc)-OH as raw materials, a first-step amide condensation was carried out in the presence of a condensing agent and an organic base to obtain an intermediate product. Subsequently, the intermediate product was deprotected from Fmoc and then subjected to a second-step amide condensation with Boc-Arg(Pbf)-OH in the presence of a condensing agent and an organic base. The reaction product was purified by extraction and silica gel column chromatography to obtain N1-Arg-OMe. The head intermediates Boc-G2K-OMe, Boc,Pbf-G2R-OMe, M1-Arg-OMe and N1-Arg-OMe were subjected to alkaline hydrolysis with NaOH in methanol solution. After adjusting the pH and extraction, the corresponding carboxylated head products Boc-G2K-OH, Boc,Pbf G2R-OH, M1-Arg-OH and N1-Arg-OH were obtained, respectively.

6. The four cationic lipopeptides according to claim 3, characterized in that, The method for preparing the cationic lipopeptide tail is as follows: N-Boc-ethylenediamine and 1-bromooctadecane were reacted in an organic solvent under alkaline conditions to generate a Boc-protected intermediate. The Boc-protected intermediate was then extracted, washed, dried, and purified by column chromatography. Under acidic conditions, the purified intermediate was deprotected from the Boc protecting group in an organic solvent. The reaction solution obtained from the deprotection reaction was neutralized, extracted, dried, and concentrated to obtain the target product C18.

7. The four cationic lipopeptides according to claim 3, characterized in that, The preparation method includes a head-tail linkage step and a deprotection step; wherein, the cationic lipopeptides G2K-C18, G2R-C18, M1-C18 and N1-C18 are obtained by amide bond condensation reaction between the cationic lipopeptide head and the hydrophobic C18 tail compound to obtain the linkage product, and then the linkage product is deprotected under acidic conditions to obtain the final product.

8. The cationic lipopeptide lung-targeting lipid nanoparticles according to claim 1, characterized in that, The lipid nanoparticles are composed of a pharmaceutically safe carrier material that can effectively encapsulate and deliver bioactive substances. Furthermore, the bioactive substance is a nucleic acid; Furthermore, the nucleic acid is any one or a combination of the following: small interfering RNA (siRNA), messenger RNA (mRNA), microRNA, circular RNA (circRNA), guide RNA (gRNA), short hairpin RNA (shRNA), long non-coding RNA (lncRNA), plasmid DNA, or antisense oligonucleotides (ASOs).

9. The cationic lipopeptide lung-targeting lipid nanoparticles according to claim 1, characterized in that, The method includes the following steps: Cationic lipopeptides, ionizable lipids, cholesterol, and PEGylated lipids were dissolved in an organic solvent to form a lipid phase, and nucleic acids were dissolved in a water-soluble buffer to form an aqueous phase. The lipid phase and the aqueous phase were mixed, and the self-assembly of lipid nanoparticles and nucleic acid encapsulation were driven by mechanical mixing, followed by static incubation. Finally, the lung-targeting lipid nanoparticles were purified by membrane filtration. Furthermore, the mechanical mixing method is achieved through vortexing, blowing, or a microfluidic device; Furthermore, the membrane filtration method is ultrafiltration.

Images