Cationic lipids, lipid nanoparticles, and uses thereof

Abstract

The application relates to the technical field of medicines, in particular to a cationic lipid, a lipid nanoparticle and application. The general structure of the cationic lipid is as follows: wherein R1 is an aminophenyl or an aminoalkyl, R2 is a saturated alkyl chain or an unsaturated alkyl chain, and n is a positive integer. The cationic lipid provided by the application is shaped into a more optimized conical structure by embedding a bulky benzene ring and skillfully constructing three tail chains. The unique design not only facilitates the endosome escape process of the lipid nanoparticle in cells, but also significantly improves the delivery efficiency of the internal drug.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to a cationic lipid, lipid nanoparticles, and their applications. Background Technology

[0002] Cancer has become a major disease posing a serious threat to human health worldwide and is one of the leading causes of death in many countries. Traditional chemotherapy and radiotherapy, as the main treatments for cancer, while able to inhibit tumor growth to some extent, have many limitations. With the continuous development of medical technology, cell-based therapy and gene therapy have gradually become research hotspots in the field of cancer treatment. Nucleic acid drugs (such as siRNA, miRNA, and mRNA) can exert therapeutic effects by inhibiting tumor growth or promoting tumor cell apoptosis due to their ability to precisely regulate gene expression. However, nucleic acid drugs face many challenges in their delivery within the body. Nucleic acid drugs themselves have poor stability and are easily degraded by nucleases in the body, causing them to lose their activity before reaching target cells. At the same time, due to the special structure and physiological environment of tumor tissue, nucleic acid drugs have difficulty penetrating tumor tissue and cannot effectively accumulate at the tumor site to exert their effects.

[0003] Lipid nanoparticles (LNPs) serve as highly efficient drug delivery carriers, protecting nucleic acid drugs from degradation by nucleases and enhancing their accumulation in tumor tissues. Currently, LNPs typically consist of four components: cationic lipids, amphiphilic phospholipids (i.e., accessory lipids), cholesterol (structural lipids), and polymeric lipids. Their particle size is usually in the range of 20-200 nanometers, a size range that enables efficient in vivo drug delivery. In the development of novel LNPs, cationic lipids have become a key research focus. Traditional permanently charged cationic lipids readily interact with negatively charged serum proteins in the blood, leading to aggregation in the blood, which shortens the half-life of LNPs and produces significant toxic side effects, greatly limiting their application as carriers. In contrast, ionizable LNPs are electrically neutral under physiological pH conditions, exhibit weaker interactions with anion exchange membranes in the blood, and possess good biocompatibility. Once LNPs enter the endosome via endocytosis, the low pH inside the endosome causes the free amino groups to protonate, creating a proton sponge effect. This positive charge generation is crucial for promoting endosome escape of LNPs. Therefore, most current research on LNP systems focuses on ionizable lipids, identifying lipid nanoparticles with high transfection efficiency by screening for optimal head groups, linker sites, and tail structures. However, the delivery efficiency of existing LNP systems in solid tumors still needs further improvement, especially under the complex conditions of the tumor microenvironment, such as high interstitial pressure, hypoxia, and abundant extracellular matrix, all of which adversely affect the delivery performance of LNPs. Summary of the Invention

[0004] In view of the shortcomings of the prior art, the present invention provides a cationic lipid, lipid nanoparticles and their applications, thereby solving the problem of low delivery efficiency of existing lipid nanoparticles.

[0005] The technical solution adopted by the present invention to solve the above-mentioned technical problems is as follows:

[0006] In a first aspect, the present invention provides a cationic lipid, the general structural formula of which is:

[0007]

[0008] Wherein, R1 is an aminophenyl or aminoalkyl, R2 is a saturated alkyl chain or an unsaturated alkyl chain, and n is a positive integer.

[0009] Preferably, R1 is an aminophenyl group, R2 is a saturated alkyl chain with a carbon chain length of 6-12, and n is a positive integer from 1 to 8.

[0010] Preferably, the cationic lipid has one of the following structural formulas:

[0011]

[0012] In a second aspect, the present invention provides a lipid nanoparticle comprising the above-described cationic lipids, auxiliary lipids, structural lipids, and polymeric lipids.

[0013] Preferably, the cationic lipids in the lipid nanoparticles account for 10-65% by mass.

[0014] Preferably, the lipid nanoparticles have a nitrogen-to-phosphorus ratio of 2-12.

[0015] Preferably, the auxiliary lipid is selected from 1,2-distearyl-sn-glycerol-3-phosphate choline, 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine, distearyl-phosphatidyl-ethanolamine, dipalmitoylphosphatidylethanolamine, dimyristoylphosphatidylethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate choline, 1,2-dimyristoyl-sn-glycerol-phosphate choline, 1,2-dioleoyl-sn-glycerol-3-phosphate choline, 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline, 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline, 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline, 1,2-dipalmitoyl-sn-glycerol-3-o-4'-(N,N,N-trimethyl)homoserine, 1,2-diundecanoyl-sn-glycerol-phosphate choline, 1-palmitoyl The lipid is selected from one or more of the following: 2-oleoyl-sn-glycerol-3-phosphocholine, sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, and phosphatidylserine. The structural lipid is selected from one or more of the following: cholesterol, steroids, cholesterol, fucosterol, squalene, ergosterol, stigmasterol, ergosterol, β-sitosterol, lanosterol, non-steroidal, brassosterol, cycloartenol, campesterol, and brassosterol. The polymeric lipid is selected from one or more of the following: distearylphosphatidylethanolamine polyethylene glycol, dimyristoylglycerol-3-methoxy polyethylene glycol, methoxy polyethylene glycol bis(tetradecylacetamide), 1,2-diacetyl-glucanol-3-methoxydimethylethylene glycol, and polyethylene glycol dicholesterol.

[0016] In a third aspect, the present invention provides a pharmaceutical formulation comprising the lipid nanoparticles and nucleic acid described above.

[0017] Preferably, the nucleic acid is selected from one or more of mRNA, siRNA, aiRNA, miRNA, dSRNA, shRNA, lncRNA, saRNA, rRNA, RNA, DNA, cDNA, plasmid, aptamer, tRNA, piRNA, circRNA, antisense oligonucleotide, ribozyme, PNA, and DNAzyme.

[0018] In a fourth aspect, the present invention provides the use of the above-described cationic lipids and / or the above-described lipid nanoparticles and / or the above-described pharmaceutical formulations in the preparation of a drug for treating solid tumors.

[0019] Beneficial effects:

[0020] This invention discloses a cationic lipid, lipid nanoparticles, and their applications. The cationic lipid provided by this invention is shaped into a more optimized conical structure by embedding a large-volume benzene ring and cleverly constructing three tail chains. This unique design not only facilitates the endosome escape process of the lipid nanoparticles within cells but also significantly improves the intracellular drug delivery efficiency. In addition, the lipid nanoparticles utilize enhanced permeability and retention (EPR) and the transcellular transport properties of their nanoparticles (NPs) to achieve targeted targeting of solid tumors. Attached Figure Description

[0021] Figure 1 This is a schematic diagram illustrating the preparation and treatment of targeted solid tumor LNPs in Example 1 and Example 6 of the present invention;

[0022] Figure 2 To prepare the cationic lipid 9 in Example 1 1 H-NMR;

[0023] Figure 3 To prepare the cationic lipid 9 in Example 1 13 C-NMR;

[0024] Figure 4 To prepare high-resolution mass spectrometry for the cationic lipid 9 in Example 1;

[0025] Figure 5 To prepare the cationic lipid 1 in Example 1 1 H-NMR;

[0026] Figure 6 To prepare the cationic lipid 1 in Example 1 13 C-NMR;

[0027] Figure 7 To prepare high-resolution mass spectrometry for cationic lipid 1 in Example 1;

[0028] Figure 8 To prepare the cationic lipid 2 in Example 1 1 H-NMR;

[0029] Figure 9 To prepare the cationic lipid 2 in Example 1 13 C-NMR;

[0030] Figure 10 To prepare high-resolution mass spectrometry for the cationic lipid 2 in Example 1;

[0031] Figure 11 To prepare the cationic lipid 3 in Example 1 1 H-NMR;

[0032] Figure 12 To prepare the cationic lipid 3 in Example 1 13 C-NMR;

[0033] Figure 13 To prepare high-resolution mass spectrometry for cationic lipid 3 in Example 1;

[0034] Figure 14 To prepare the cationic lipid 4 in Example 1 1 H-NMR;

[0035] Figure 15 To prepare the cationic lipid 4 in Example 1 13 C-NMR;

[0036] Figure 16 To prepare high-resolution mass spectrometry for cationic lipid 4 in Example 1;

[0037] Figure 17 To prepare the cationic lipid 5 in Example 1 1 H-NMR;

[0038] Figure 18 To prepare the cationic lipid 5 in Example 1 13 C-NMR;

[0039] Figure 19 To prepare high-resolution mass spectrometry for the cationic lipid 5 in Example 1;

[0040] Figure 20 To prepare the cationic lipid 6 in Example 1 1 H-NMR;

[0041] Figure 21 To prepare the cationic lipid 6 in Example 1 13 C-NMR;

[0042] Figure 22 To prepare high-resolution mass spectrometry for the cationic lipid 6 in Example 1;

[0043] Figure 23 To prepare the cationic lipid 7 in Example 1 1 H-NMR;

[0044] Figure 24 To prepare the cationic lipid 7 in Example 1 13 C-NMR;

[0045] Figure 25 To prepare high-resolution mass spectrometry for the cationic lipid 7 in Example 1;

[0046] Figure 26 To prepare the cationic lipid 8 in Example 1 1 H-NMR;

[0047] Figure 27 To prepare the cationic lipid 8 in Example 1 13 C-NMR;

[0048] Figure 28 To prepare high-resolution mass spectrometry for cationic lipid 8 in Example 1;

[0049] Figure 29 The particle size, PDI, and Zeta potential of the LNP in Example 1 of this invention;

[0050] Figure 30 This describes the transfection status of Fluc mRNA LNP loaded in Example 2 of the present invention;

[0051] Figure 31 This document describes the transfection results of Flux mRNA9-LNP loaded at different nucleic acid concentrations in Example 2 of this invention.

[0052] Figure 32 This describes the in vivo transfection of Fluc mRNA LNP loaded with LNP via subcutaneous injection in Example 3 of the present invention.

[0053] Figure 33 This describes the in vivo transfection of SM-102, 3, and 9-Fluc mRNA LNPs via intravenous injection in Example 3 of this invention.

[0054] Figure 34 This document shows the organ distribution of SM-102, 3, and 9-Fluc mRNA LNPs transfected in vivo via intravenous injection in Example 3 of this invention; and the average fluorescence intensity of the three liposomes in the liver and spleen, the main distribution organs.

[0055] Figure 35 This is an in vivo imaging of mice with tumors after intravenous injection of CY5-Sirna LNP in Example 4 of the present invention.

[0056] Figure 36 This describes the accumulation of CY5-Sirna LNP-loaded material in tumor tissue via intravenous injection in Example 4 of the present invention.

[0057] Figure 37 The particle size, PDI, Zeta potential, and TEM of the most preferred lipid nanoparticles in Example 5 of the present invention were measured over 24 days.

[0058] Figure 38 The biosafety (hemolysis rate and cytotoxicity) of the most preferred lipid nanoparticles in Example 5 of this invention was determined.

[0059] Figure 39 The final tumor volume after treatment in the PBS group, Trastuzumab group, Arry-380 group, and 9-LNP-mrna group in Example 6 of this invention;

[0060] Figure 40 The figures show the tumor growth inhibition curves and mouse weight changes during treatment in the PBS group, Trastuzumab group, Arry-380 group, and 9-LNP-mrna group in Example 6 of this invention. Detailed Implementation

[0061] This invention provides a cationic lipid, lipid nanoparticles, and their applications. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0062] This invention provides a cationic lipid, the general structural formula of which is:

[0063]

[0064] Wherein, R1 is an aminophenyl or aminoalkyl, R2 is a saturated alkyl chain or an unsaturated alkyl chain, and n is a positive integer.

[0065] The cationic lipids provided in this invention are shaped into a more optimized conical structure by embedding a large benzene ring and cleverly constructing three tail chains. This unique design not only facilitates the endosome escape process of lipid nanoparticles within cells but also significantly improves the delivery efficiency of drugs within them.

[0066] In some embodiments, R1 is an aminophenyl group, R2 is a saturated alkyl chain with a carbon chain length of 6-12, and n is a positive integer from 1 to 8.

[0067] Under these constraints, the introduction of aminophenyl groups significantly enhances the structural rigidity of cationic lipids, thus prolonging their circulation time in vivo and promoting endosome membrane fusion through a rigid framework, thereby enhancing their endosome escape capability. Meanwhile, changes in the length of the linking carbon chains affect the thickness and compactness of the cationic lipid bilayer. At this length, the cationic lipid bilayer can be made more compact, improving its stability and reducing nucleic acid degradation in the bloodstream.

[0068] In some embodiments, the cationic lipid has one of the following structural formulas:

[0069]

[0070] This invention provides a method for preparing the above-mentioned cationic lipid 9, the reaction route of which is shown below. The preparation method includes the following steps:

[0071]

[0072] S1. Sodium hydroxide, pentaerythritol, 1-bromononane and tetrabutylammonium bromide are mixed and reacted to obtain compound a;

[0073] In the reaction, pentaerythritol acts as a nucleophile, with its hydroxyl group participating in a nucleophilic substitution reaction, reacting with 1-bromononane to generate the corresponding alkyl ether; 1-bromononane acts as an electrophile, with its bromine atom potentially being replaced by the hydroxyl group of pentaerythritol to form an alkyl ether; tetrabutylammonium bromide is a phase-transfer catalyst, its role being to accelerate the reaction by forming ion pairs with the reactants, lowering the activation energy of the reaction, thereby promoting the reaction; sodium hydroxide is a strong base, acting as a basic catalyst in the reaction, providing an alkaline environment to promote the deprotonation of the hydroxyl group of pentaerythritol, enhancing its nucleophilicity, and thus accelerating the reaction.

[0074] S2. Compound a is mixed with triethylamine, 4-dimethylaminopyridine (DMAP), anhydrous dichloromethane, and 3-bromopropionyl chloride, and reacted to obtain compound b.

[0075] In the reaction, compound a, containing a hydroxyl functional group, acts as the substrate and undergoes an acylation reaction with 3-bromopropionyl chloride to form an ester bond. Triethylamine acts as a basic catalyst, neutralizing acidic byproducts (such as HCl) generated during the reaction, maintaining a neutral environment in the reaction system, and promoting the smooth progress of the reaction. DMAP, as a highly efficient catalyst, can significantly improve the rate and yield of the acylation reaction. DMAP accelerates acyl transfer by enhancing the nucleophilicity of the reaction system, especially showing excellent performance in reaction substrates with large steric hindrance. 3-bromopropionyl chloride acts as the acylation reagent, providing an acyl group (-CO-) that reacts with the hydroxyl group in compound a to form an ester bond. Dichloromethane acts as the reaction solvent, providing a suitable environment for the reaction to proceed, while ensuring good solubility of the reactants and catalyst.

[0076] S3. Compound b is mixed with 2-(methylamino)-1-phenylethanol (α-(methylaminomethyl)benzyl alcohol), potassium carbonate and acetonitrile, and reacted to obtain compound 9.

[0077] In the reaction, compound b serves as the substrate and contains a bromine atom. 2-(methylamino)-1-phenylethanol (α-(methylaminomethyl)benzyl alcohol) acts as a nucleophile, with its secondary amino group (-NH-) participating in the reaction and undergoing a nucleophilic substitution reaction with the bromine atom in compound b to form a tertiary amine. Potassium carbonate acts as a basic reagent to neutralize acidic byproducts (such as HCl) generated in the reaction, maintaining an alkaline environment in the reaction system and promoting the smooth progress of the reaction. Acetonitrile acts as the reaction solvent, providing a suitable polar aprotic environment for the reaction to proceed, ensuring good solubility of the reactants and catalyst, and also facilitating the reaction.

[0078] In some embodiments, the molar ratio of sodium hydroxide, pentaerythritol, 1-bromononane, and tetrabutylammonium bromide is 1:(0.2-0.5):(0.8-1.2):(0.025-0.075). More preferably, the molar ratio of sodium hydroxide, pentaerythritol, 1-bromononane, and tetrabutylammonium bromide is 1:(0.2-0.4):(0.8-1.0):(0.025-0.05). Even more preferably, the molar ratio of sodium hydroxide, pentaerythritol, 1-bromononane, and tetrabutylammonium bromide is 1:0.3:0.966:0.0375.

[0079] In some embodiments, the reaction temperature in step S1 is 65-90°C. More preferably, the reaction temperature is 75-90°C. Even more preferably, the reaction temperature is 80°C.

[0080] In some embodiments, the reaction time in step S1 is 4-8 hours. More preferably, the reaction time is 5-7 hours. Even more preferably, the reaction time is 6 hours.

[0081] In some embodiments, after step S1, there are further processes of washing, rotary evaporation of the organic phase, and purification.

[0082] In some embodiments, the washing process includes water washing, acid washing, and water washing in sequence.

[0083] In some embodiments, the water washing uses NaCl solution, and the acid washing uses HCl solution.

[0084] In some embodiments, the molar ratio of compound a to triethylamine, 4-dimethylaminopyridine, anhydrous dichloromethane, and 5-bromopentanoyl chloride is 1:(0.1-0.5):(0.04-0.09):(18-25):(0.2-0.8). More preferably, the molar ratio of compound a to triethylamine, 4-dimethylaminopyridine, anhydrous dichloromethane, and 5-bromopentanoyl chloride is 1:(0.1-0.3):(0.05-0.08):(19-24):(0.3-0.7). Even more preferably, the molar ratio of compound a to triethylamine, 4-dimethylaminopyridine, anhydrous dichloromethane, and 5-bromopentanoyl chloride is 1:0.196:0.064:21.277:0.511.

[0085] In some embodiments, the reaction temperature in step S2 is 15-30°C. More preferably, the reaction temperature is 15-20°C. Even more preferably, the reaction temperature is 23°C.

[0086] In some embodiments, the reaction time in step S2 is 1-4 hours. More preferably, the reaction time is 2-3 hours. Even more preferably, the reaction time is 2 hours.

[0087] In some embodiments, after step S2, there are further processes of washing, rotary evaporation of the organic phase, and purification.

[0088] In some embodiments, the washing process includes water washing, acid washing, and water washing in sequence.

[0089] In some embodiments, the water washing uses NaCl solution, and the acid washing uses HCl solution.

[0090] In some embodiments, the molar ratio of compound b to 2-(methylamino)-1-phenylethanol (α-(methylaminomethyl)benzyl alcohol), potassium carbonate, and acetonitrile is 1:(0.2-0.5):(0.1-0.4):(30-50). More preferably, the molar ratio of compound b to 2-(methylamino)-1-phenylethanol (α-(methylaminomethyl)benzyl alcohol), potassium carbonate, and acetonitrile is 1:(0.3-0.4):(0.2-0.3):(40-50). Even more preferably, the molar ratio of compound b to 2-(methylamino)-1-phenylethanol (α-(methylaminomethyl)benzyl alcohol), potassium carbonate, and acetonitrile is 1:0.347:0.255:42.553.

[0091] In some embodiments, the reaction temperature in step S3 is 0-5°C. More preferably, the reaction temperature is 0-2°C. Even more preferably, the reaction temperature is 0°C.

[0092] In some embodiments, the reaction time in step S3 is 1-4 hours. More preferably, the reaction time is 2-3 hours. Even more preferably, the reaction time is 2 hours.

[0093] In some embodiments, after step S3, there are further processes of washing, rotary evaporation of the organic phase, and purification.

[0094] In some embodiments, the washing process includes water washing, acid washing, and water washing in sequence.

[0095] In some embodiments, the water washing uses NaCl solution, and the acid washing uses HCl solution.

[0096] This invention provides a lipid nanoparticle, which includes the above-mentioned cationic lipids, auxiliary lipids, structural lipids, and polymeric lipids.

[0097] In some embodiments, the auxiliary lipid is selected from 1,2-distearyl-sn-glycerol-3-phosphate choline, 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine, distearyl-phosphatidyl-ethanolamine, dipalmitoylphosphatidylethanolamine, dimyristoylphosphatidylethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate choline, 1,2-dimyristoyl-sn-glycerol-phosphate choline, 1,2-dioleoyl-sn-glycerol-3-phosphate choline, 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline, 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline, 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline, 1,2-dipalmitoyl-sn-glycerol-3-o-4'-(N,N,N-trimethyl)homoserine, 1,2-diundecanoyl-sn-glycerol-phosphate choline, 1- The lipid structure is selected from one or more of palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine, sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, and phosphatidylserine. The structural lipid is selected from one or more of cholesterol, steroids, cholesterol, fucosterol, squalene, ergosterol, stigmasterol, ergosterol, β-sitosterol, lanosterol, non-steroidal, brassosterol, cycloartenol, campesterol, and brassosterol. The polymer lipid is selected from one or more of distearylphosphatidylethanolamine polyethylene glycol, dimyristoylglycerol-3-methoxy polyethylene glycol, methoxy polyethylene glycol bistetradecylacetamide, 1,2-diacetyl-glucanol-3-methoxydimethylethylene glycol, and polyethylene glycol dicholesterol.

[0098] This invention provides a pharmaceutical formulation comprising the lipid nanoparticles and nucleic acid described above.

[0099] In some embodiments, the nucleic acid is selected from one or more of mRNA, siRNA, aiRNA, miRNA, dSRNA, shRNA, lncRNA, saRNA, rRNA, RNA, DNA, cDNA, plasmid, aptamer, tRNA, piRNA, circRNA, antisense oligonucleotide, ribozyme, PNA, and DNAzyme.

[0100] In some embodiments, the encoding of the light and heavy chains of the trastuzumab mRNA is provided.

[0101] This invention utilizes carefully designed lipid nanoparticles to deliver mRNA encoding trastuzumab. This method effectively reduces off-target effects and degradation in the circulatory system, ensuring accurate transfection of the mRNA into solid tumors. Once inside tumor cells, the mRNA directs in situ production of trastuzumab, achieving precise killing from within. This therapeutic strategy significantly increases drug concentration at the tumor site while drastically reducing the drug dose in systemic circulation, thereby minimizing damage to healthy tissues.

[0102] This invention provides a method for preparing the above-mentioned pharmaceutical formulation, the method comprising the following steps:

[0103] (1) Dissolve cationic lipids, auxiliary lipids, structural lipids and polymer lipids in an organic solvent in a certain proportion to form a lipid mixture.

[0104] (2) Dissolve the nucleic acid in a buffer solution to obtain a nucleic acid solution.

[0105] (3) Using microfluidic technology, the lipid mixture obtained in step (1) is mixed with the nucleic acid solution obtained in step (2), and the organic solvent is removed by dialysis or ultrafiltration to form a drug formulation.

[0106] In some embodiments, in step (1), the molar ratio of the cationic lipid, auxiliary lipid, structural lipid, and polymeric lipid is (30-65):(6-15):(20-50):(0.3-3.5). Alternatively, the molar ratio of the cationic lipid, auxiliary lipid, structural lipid, and polymeric lipid can be (40-60):(8-12):(30-45):(0.5-3). Or, the molar ratio of the cationic lipid, auxiliary lipid, structural lipid, and polymeric lipid can be 50:10:38.5:1.5.

[0107] In some embodiments, in step (2), sodium hydroxide, pentaerythritol, 1-bromononane and tetrabutylammonium bromide are mixed and reacted to obtain compound 1, which is then mixed with mRNA buffer (selected from one or more of 25mM, 50mM and 100mM citrate-sodium citrate buffer, preferably 50mM citrate-sodium citrate buffer) via a staggered-herringbone microfluidic system (SHM).

[0108] In some implementations, in step (2), the total flow rate (TFR) is 10-30 mL / min. The total flow rate (TFR) can be 12-22 mL / min, or 16 mL / min.

[0109] In some implementations, in step (2), the flow rate ratio (FRR) is 1:(2-5), the flow rate ratio (FRR) can be 1:(2-4), or the flow rate ratio (FRR) can be 1:3.

[0110] In some implementations, step (2) is followed by a process to remove ethanol and unloaded RNA.

[0111] In some embodiments, the process of removing ethanol and unloaded RNA is performed using dialysis.

[0112] In some embodiments, the process of removing ethanol and unloaded RNA is performed using dialysis, specifically: a dialysis bag with a molecular weight cutoff of 15 kDa is used, and dialysis is performed in PBS buffer solution, with the solution changed every 2-4 hours, and the operation is repeated until dialysis is completed after 18-24 hours.

[0113] In some embodiments, the dialysis procedure is performed in PBS buffer solution, with the solution changed every 2 hours, and the procedure is repeated until the dialysis is completed after 18 hours.

[0114] In some embodiments, the process of removing ethanol and unloaded RNA is performed using ultrafiltration.

[0115] In some embodiments, the process of removing ethanol and unloaded RNA is carried out using an ultrafiltration method, specifically using an ultrafiltration tube with a molecular weight cutoff of 10-100 kDa.

[0116] In some implementations, an ultrafiltration tube with a molecular weight cutoff of 50 kDa is selected.

[0117] In some embodiments, after the pharmaceutical preparation obtained in step (3) is collected, it is diluted with 1-3 times the volume of citrate buffer or acetate buffer.

[0118] In some implementations, the solution is diluted with one volume of citrate buffer.

[0119] In some embodiments, the diluted pharmaceutical preparation is subjected to centrifugation and ultrafiltration at 1500-3500g to 0.2-0.5 times its original volume.

[0120] In some implementations, the material is subjected to 3000g centrifugation and ultrafiltration to 0.25 times its original volume.

[0121] In some embodiments, PBS buffer is added to the solution after centrifugation and ultrafiltration to the original volume, and then centrifuged and ultrafiltered again to 0.25 times the original volume. This process is repeated 2-5 times to reduce the ethanol content to below 0.1-0.9%.

[0122] In some implementations, the step is repeated three times to reduce the ethanol content to below 0.5%.

[0123] In some implementations, the final concentration is achieved by ultrafiltration using a buffer solution containing sucrose preservative.

[0124] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are merely some embodiments of the present invention, not all embodiments, and are intended only to illustrate the present invention and not to limit it. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0125] Prepare the following nine cationic lipid structures:

[0126]

[0127] Preparation Example 1: A schematic diagram of its preparation is shown below. Figure 1

[0128]

[0129] Synthetic route of cationic lipid 9

[0130] Pentaerythritol (2.2 g, 16.1 mmol, 1 equiv) was added to freshly prepared sodium hydroxide solution (30 g NaOH and 30 mL water), and stirred at 80 °C for 1 hour. Subsequently, 1-bromononane (10 g, 48.3 mmol, 3 equiv) and tetrabutylammonium bromide (0.65 g, 2.01 mmol, 0.125 equiv) were added. After the reaction was complete, the target compound a (1.32 g, 15.94% yield) was obtained through conventional post-treatment steps. 1 H NMR (400MHz, CDCl3) δ3.70 (s, 2H), 3.44 (s, 6H), 3.38 (t, J = 6.5Hz, 6H), 1.54 (p, J = 6.6Hz, 6H), 1.36-1.22 (m, 36H), 0.92-0.84 (m, 9H).

[0131] The reaction was carried out using compound a (0.47 g, 0.92 mmol, 1 equiv), triethylamine (NEt3, 92 mg, 0.92 mmol, 1 equiv), 4-dimethylaminopyridine (22 mg, 0.184 mmol, 0.2 equiv) and 5-bromopentanoyl chloride (0.24 g, 1.2 mmol, 1.3 equiv) as starting materials, and finally the target compound b (0.49 g, yield 79.03%) was obtained as a colorless oil. 1 H NMR (400MHz, CDCl3) δ4.11(s,2H),3.41(t,J=6.6Hz,2H),3.38(s,12H),2.34(t,J=7.3Hz,2H),1.95-1.85(m,2 H),1.78(dtd,J=15.0,8.2,2.6Hz,2H),1.51(p,J=6.6Hz,6H),1.35-1.20(m,36H),0.88(td,J=7.0,1.4Hz,9H).

[0132] Compound b (0.49 g, 0.72 mmol, 1 equiv), α-(methylaminomethyl)benzyl alcohol (0.17 g, 1.08 mmol, 1.5 equiv), potassium carbonate (K₂CO₃, 0.12 g, 0.86 mmol, 1.2 equiv), and 20 mL of acetonitrile were mixed. The reaction mixture was heated to 95 °C and stirred continuously for 3 hours. After the reaction was complete, the mixture was cooled to 23 °C, and the acetonitrile was removed under reduced pressure. Then, 20 mL of water was added, and the mixture was extracted three times with 20 mL of dichloromethane. The resulting organic phases were combined and dried with anhydrous magnesium sulfate. After filtration, the filtrate was concentrated to approximately 3 mL and purified by column chromatography (using SiO₂ as the stationary phase and dichloromethane / methanol = 50 / 1 to 20 / 1 as the eluent) to obtain an oily target compound, yielding a colorless oily target compound 9 (0.41 g, yield 75.93%). 1H NMR (400MHz, CDCl3) δ7.40-7.32(m,4H),7.31-7.27(m,1H),4.84(s,1H),4.11(s,2H),3.42-3.28(m,12H),2.61(s,4H),2. 48(s,3H),2.39-2.29(m,2H),1.68(d,J=9.8Hz,5H),1.50(d,J=6.9Hz,6H),1.35-1.16(m,36H),0.88(t,J=6.9Hz,9H).13C NMR(101MHz, CDCl3)δ128.46,125.83,77.22,71.63,69.47,44.46,31.93,29.64,29.53,29.34,26.20,22.70,14.13.HRMS[M+H + ]: C 46 H 85 Calculated value of NO6: 748.1840; Found value: 748.6498.

[0133] cationic lipid 9 1 H-NMR, 13 C-NMR and high-resolution mass spectrometry are shown in [link to C-NMR and high-resolution mass spectrometry]. Figure 2-4 .

[0134] Based on the synthesis method of cationic lipid 9, the synthesis methods of the other cationic lipids numbered 1-8 are similar:

[0135] cationic lipid 1

[0136] The intermediate (0.40 g, 0.71 mmol, 1 equiv), α-(methylaminomethyl)benzyl alcohol (0.16 g, 1.06 mmol, 1.5 equiv), potassium carbonate (K2CO3, 0.12 g, 0.87 mmol, 1.2 equiv), and 20 mL of acetonitrile were mixed and reacted according to a procedure similar to that used in the synthesis of cationic lipid 9 to give the target compound in an oily form (0.32 g, yield 71.11%). 1H NMR (400MHz, CDCl3) δ7.43-7.33(m,4H),7.32-7.26(m,1H),4.81(s,1H),4.19(s,2H),3.42-3.33(m,12H),3.01( s,1H),2.84(s,1H),2.60(s,4H),2.45(s,3H),1.54(q,J=6.8Hz,6H),1.37-1.26(m,24H),0.90(t,J=7.0Hz,9H). 13C NMR (101MHz, CDCl3) δ128.44,125.84,77.22,71.64,69.49,44.44,31.89,29.63,29.18,26.15,22.66,14.12.HRMS[M+H + ]: Calculated value of C38H69NO6: 635.9710; Found value: 636.5209.

[0137] cationic lipid 2

[0138] Using intermediates (0.4 g, 0.69 mmol, 1 equiv), α-(methylaminomethyl)benzyl alcohol (0.16 g, 1.04 mmol, 1.5 equiv), potassium carbonate (K2CO3, 0.12 g, 0.83 mmol, 1.2 equiv), and 20 mL of acetonitrile (MeCN) as starting materials, the reaction was carried out in a manner similar to that used in the synthesis of cationic lipid 9, yielding the target compound (0.36 g, 80% yield) in a colorless oil. 1 HNMR(400MHz, CDCl3)δ7.43-7.33(m,4H),7.33-7.26(m,1H),4.80(s,1H),4.15(s,2H),3.41-3.34(m,12H),2.70(s, 1H),2.59(s,3H),2.48-2.36(m,5H),1.92(s,2H),1.54(t,J=6.9Hz,6H),1.37-1.26(m,24H),0.90(t,J=6.8Hz,9H). 13 C NMR (101MHz, CDCl3) δ128.47,125.83,77.23,71.63,69.46,44.45,31.88,29.63,29.17,26.15,22.66,14.12.HRMS[M+H + ]: C 39 H 71 Calculated value of NO6: 649.9980; Found value: 650.5349.

[0139] cationic lipid 3

[0140] Using the intermediate product (0.6 g, 1.01 mmol, 1 equiv), α-(methylaminomethyl)benzyl alcohol (0.23 g, 1.52 mmol, 1.5 equiv), potassium carbonate (K2CO3, 0.17 g, 1.21 mmol, 1.2 equiv), and 20 mL of acetonitrile (MeCN) as starting materials, the reaction was carried out in a similar manner to that of cationic lipid 9 to obtain the target compound (0.49 g, yield 73.13%), which was colorless and oily.1 HNMR(400MHz, CDCl3)δ7.40-7.31(m,4H),7.31-7.23(m,1H),4.76(s,1H),4.12(s,2H),3.39-3.32(m,12H),2.64(s,1H),2.54 (s,3H),2.41(s,3H),2.34(t,J=7.2Hz,2H),1.72-1.62(m,4H),1.51(p,J=6.7Hz,4H),1.35-1.20(m,26H),0.91-0.85(m,9H). 13 C NMR (101MHz, CDCl3) δ128.45,125.83,77.23,71.62,69.47,44.45,31.89,29.63,29.18,26.15,22.66,14.12.HRMS[M+H+]: C 40 H 73 Calculated value of NO6: 664.0250; Found value: 664.5496.

[0141] cationic lipid 4

[0142] Using the intermediate product (0.54 g, 0.89 mmol, 1 equiv), α-(methylaminomethyl)benzyl alcohol (0.2 g, 1.34 mmol, 1.5 equiv), potassium carbonate (K2CO3, 0.15 g, 1.07 mmol, 1.2 equiv), and 20 mL of acetonitrile (MeCN) as starting materials, the reaction was carried out in a manner similar to that used in the synthesis of cationic lipid 9, yielding the target compound (0.46 g, yield 76.67%) as a colorless oil. 1 H NMR (400MHz, CDCl3) δ7.43-7.33(m,4H),7.33-7.26(m,1H),4.81(s,1H),4.18(s,2H),3.44-3.35(m,12H),3.03( s,1H),2.86(s,1H),2.61(s,4H),2.46(s,3H),1.54(t,J=7.0Hz,6H),1.37-1.24(m,30H),0.90(t,J=6.9Hz,9H). 13 C NMR (101MHz, CDCl3) δ125.84,71.65,69.48,44.44,31.89,29.63,29.48,29.34,26.20,22.69,14.12.HRMS[M+H+]: C 41 H 75 Calculated value of NO6: 678.0520; Found value: 678.5692.

[0143] cationic lipid 5

[0144] Using compound 4-8C (0.65 g, 1.05 mmol, 1 equiv), α-(methylaminomethyl)benzyl alcohol (0.24 g, 1.58 mmol, 1.5 equiv), potassium carbonate (K2CO3, 0.17 g, 1.26 mmol, 1.2 equiv), and 20 mL of acetonitrile (MeCN) as starting materials, the reaction was carried out in a similar manner to that of cationic lipid 9 to obtain the target compound (0.55 g, yield 75.34%), which was a colorless oil. 1 H NMR (400MHz, CDCl3) δ7.40-7.31(m,4H),7.30-7.27(m,1H),4.79(s,1H),4.13(s,2H),3.40-3.31(m,12H),2.69(s,1H),2 .58(s,3H),2.45-2.35(m,5H),1.90(s,2H),1.51(p,J=6.6Hz,6H),1.28(dd,J=11.2,6.0Hz,30H),0.88(t,J=6.9Hz,9H). 13 C NMR (101MHz, CDCl3) δ128.45,125.83,77.22,71.64,69.46,44.45,31.89,29.63,29.48,29.34,26.20,22.69,14.12.HRMS[M+H+]: C 42 H 77 Calculated value of NO6: 692.0790; Found value: 692.5827.

[0145] cationic lipid 6

[0146] Using the intermediate product (0.78 g, 1.23 mmol, 1 equiv), α-(methylaminomethyl)benzyl alcohol (0.28 g, 1.85 mmol, 1.5 equiv), potassium carbonate (K2CO3, 0.2 g, 1.48 mmol, 1.2 equiv), and 20 mL of acetonitrile (MeCN) as starting materials, the reaction was carried out in a manner similar to that used in the synthesis of cationic lipid 9, yielding a colorless oily target compound (0.63 g, yield 73.26%). 1H NMR (400MHz, CDCl3) δ7.40-7.31(m,4H),7.28(q,J=2.3Hz,1H),4.78(s,1H),4.12(s,2H),3.39-3.32(m,12H),2.65(s,1H),2.55 (s,2H),2.42(s,3H),2.34(t,J=7.1Hz,2H),1.72-1.62(m,5H),1.51(p,J=6.5Hz,6H),1.34-1.20(m,30H),0.88(t,J=6.9Hz,9H). 13 CNMR (101MHz, CDCl3) δ128.40,125.83,71.63,69.48,69.14,64.15,57.51,44.4 6,41.72,34.00,31.89,29.63,29.48,29.34,26.20,22.69,14.13.HRMS[M+H+]:C 43 H 79 Calculated value of NO6: 706.1060; Found value: 706.6036.

[0147] cationic lipid 7

[0148] Using the intermediate (0.44 g, 0.68 mmol, 1 equiv), α-(methylaminomethyl)benzyl alcohol (0.15 g, 1.02 mmol, 1.5 equiv), potassium carbonate (K2CO3, 0.11 g, 0.82 mmol, 1.2 equiv), and 20 mL of acetonitrile (MeCN) as starting materials, the reaction was carried out in a similar manner to that of cationic lipid 9 to obtain the target compound (0.33 g, yield 67.35%), which was a colorless oil. 1 H NMR (400MHz, CDCl3) δ7.41-7.31(m,4H),7.26(s,1H),4.78(s,1H),4.15(s,2H),3.41-3.32(m,12H),3.00(s,1H) ),2.82(s,1H),2.58(s,4H),2.43(s,3H),1.51(q,J=6.8Hz,6H),1.27(d,J=8.3Hz,36H),0.88(t,J=6.8Hz,9H). 13 C NMR (101MHz, CDCl3) δ128.40,125.85,71.65,69.50,65.90,53.05,44.45,41.80,31.93,29.64,29.53,29.34,26.20,22.70,14.13.HRMS[M+H+]: C 44 H81 Calculated value of NO6: 720.1330; Found value: 720.6198.

[0149] Cationic lipid 8

[0150] Using the intermediate (0.46 g, 0.69 mmol, 1 equiv), α-(methylaminomethyl)benzyl alcohol (0.16 g, 1.04 mmol, 1.5 equiv), potassium carbonate (K2CO3, 0.11 g, 0.83 mmol, 1.2 equiv), and 20 mL of acetonitrile (MeCN) as starting materials, the reaction was carried out in a similar manner to that of cationic lipid 9 to obtain the target compound (0.37 g, yield 72.55%), which was a colorless oil. 1 H NMR (400MHz, CDCl3) δ7.40-7.31(m,4H),7.30-7.27(m,1H),4.79(s,1H),4.13(s,2H),3.41-3.28(m,12H),2. 72-2.35(m,7H),1.90(s,2H),1.62(s,2H),1.50(q,J=6.8Hz,6H),1.35-1.15(m,36H),0.88(t,J=6.8Hz,9H). 13 C NMR 101MHz, CDCl3) δ128.44,125.83,77.23,71.64,69.46,44.45,31.93,29.64,29.53,29.34,26.20,22.70,14.13.HRMS[M+H+]: C 45 H 83 Calculated value of NO6: 734.1600; Found value: 734.6317.

[0151] The cationic lipids 1-8 prepared above 1 H-NMR, 13 C-NMR and high-resolution mass spectrometry are shown in [link to C-NMR and high-resolution mass spectrometry]. Figure 5-28 .

[0152] Preparation of the drug formulation: The cationic lipids 1-9 prepared above were mixed with DSPC, cholesterol, and DMG-PEG2000 in a ratio of 50:38.5:10:1.5 and dissolved in ethanol to obtain nine lipid nanoparticle solutions. mRNA was dissolved in citrate buffer to obtain an mRNA solution. The lipid nanoparticle solutions and mRNA solutions were mixed using a microfluidic device to form LNP-mRNA complexes. Ethanol was removed by dialysis to obtain the final drug formulation (LNP-mRNA formulation).

[0153] Example 1: Detection of particle size, potential and polydispersity index of lipid nanoparticles

[0154] To determine the particle size, zeta potential, and polydispersity index (PDI) of the lipid nanoparticles (LNPs) prepared in Preparation Example 1, dynamic light scattering (DLS) was employed using a Zetasizer Lab-Blue particle size analyzer manufactured by Malvern Instruments. This instrument is equipped with a 4mW He-Ne laser (wavelength 633nm) and an avalanche photodiode detector located at 175° of the beam, along with a temperature-controlled sample cell. Instrument parameters and measurement time were automatically set. Approximately 0.2 mL of the lipid nanoparticle solution was placed in a semi-micro sample cell (1.6 mL, polystyrene, 10 × 10 × 45 mm, Greiner Bio-One), and the volume was increased to 1 mL with deionized water. The experiment was conducted at 25°C.

[0155] Depend on Figure 29 It can be seen that the particle size of the nine LNPs prepared above is within 50-100 nm, and the PDI is generally less than 0.3. Among them, the PDI of 3-LNP, 5-LNP, and 9-LNP is less than 0.1, which is an ideal situation. The potentials of the nine LNPs are between -10 mV and +10 mV, close to neutral. Therefore, the characterization of these nine LNPs meets the expected requirements, providing conditions for subsequent experiments.

[0156] Example 2: In vitro transfection of LNP (drug formulation) loaded with Fluc-mRNA

[0157] In vitro transfection experiment: Cells were cultured to the logarithmic growth phase and then digested with trypsin. Next, the cell suspension was quantitatively transferred to 96-well plates, with 100 μL added to each well to ensure approximately 6000 cells per well. The plates were gently shaken to distribute the cells evenly, and then incubated in a constant-temperature cell culture incubator for 12 hours. After incubation, the culture medium was removed, and 500 ng of the prepared Fluc-mRNA-loaded LNPs were added to each well for further incubation. After 24 hours of incubation, firefly luciferase substrate was added to each well. After 5 minutes of incubation, the chemiluminescence intensity was measured using a multi-mode microplate reader. The group with the best transfection effect was selected to investigate the relationship between the nucleic acid content of the Fluc-mRNA-loaded LNPs and the chemiluminescence intensity.

[0158] Depend on Figure 30 , 31It was found that the chemiluminescence intensity of 9-LNP differed significantly from that of the other eight LNPs in the well plate. This indicates that changes in the linkage and tail chain length in cationic lipids have a significant impact on the in vitro transfection of LNPs. Next, the study investigated whether the chemiluminescence intensity of LNPs increased proportionally with different nucleic acid concentrations, using 9-LNP, which showed the best in vitro transfection performance in the previous experiments.

[0159] Example 3: In vivo transfection of LNPs loaded with Fluc-mRNA

[0160] Balb / c mice were used for IVIS imaging experiments. LNPs loaded with FLuc mRNA were administered intravenously / subcutaneously at a dose of 2 mg / kg. Six hours after LNP administration, mice were intraperitoneally injected with D-luciferin substrate, and fluorescence imaging was performed on the IVIS-Lumina II imaging system. The mice were then euthanized, and their major organs were dissected and subjected to fluorescence imaging on the IVIS-Lumina II imaging system.

[0161] Depend on Figure 32 It was found that 9-LNP exhibited the highest chemiluminescence intensity, followed by 3-LNP, indicating that both LNPs are suitable for in vivo transfection. To further investigate the transfection effect after in vivo circulation, 3-LNP and 9-LNP loaded with Fluc-mRNA were injected via the tail vein. After 6 hours of in vivo circulation, mouse imaging and chemiluminescence imaging after separation of major organs were performed. In this experiment, the commercially available ionizable lipid SM-102 was added for comparison.

[0162] Depend on Figure 33 , 34 It can be seen that after systemic circulation, 3-LNP and 9-LNP still have good transfection effects. When the same concentration of LNP loaded with Fluc mRNA is injected, its luminescence intensity is comparable to or even exceeds that of commercially available ionizable lipid SM-102, and it is mainly concentrated in the liver and spleen, which is consistent with the above experimental conclusions on in vivo accumulation.

[0163] Example 4: Establishment of a solid tumor model and detection of its accumulation at the solid tumor site.

[0164] 4T1 cells were collected by digestion, and after adding Matrigel, 4T1 cells (1×10⁶) were separated. 6LNPs loaded with Cy5-siRNA were implanted subcutaneously into BALB / c mice (6-8 weeks old). Seven days after growth, LNPs were injected intravenously at a dose of 2 mg / kg. Four hours after injection, imaging was performed on an IVIS-Lumina II imaging system with excitation at 649 nm and emission at 670 nm to verify the accumulation of LNPs in tumor tissue. The mice were then euthanized, their tumors were isolated, and imaged on the IVIS-Lumina II imaging system.

[0165] Depend on Figure 35 It can be seen that among all LNPs loaded with CY5-siRNA, 3-LNP and 9-LNP showed relatively obvious tumor aggregation effects, while the tumor aggregation effects of other LNPs were weaker and some did not even aggregate at the tumor site.

[0166] Tumors from each group were isolated and compared using fluorescence imaging.

[0167] Depend on Figure 36 It is evident that, in the imaging results of the separated solid tumors, 3-LNP and 9-LNP still exhibited better tumor aggregation effects, consistent with the previous in vivo imaging results of solid tumors in mice. Based on the previous characterization experiments of LNP particle size, polydispersity index, and potential, 3-LNP and 9-LNP are also closer to the ideal conditions for lipid nanoparticles, suggesting that they may have a stronger EPR effect, enabling LNPs to leak from the bloodstream and enter the tumor.

[0168] Example 5: Detection of the stability and biosafety of the optimal LNP

[0169] Similar to the method in Example 1, the particle size, potential, and polydispersity index of the optimal lipid nanoparticles were continuously monitored for 24 days. Then, the hemolysis rate and cytotoxicity of the optimal lipid nanoparticles were determined.

[0170] Depend on Figure 37 , 38 It can be seen that 9-LNP can maintain its stability well for nearly a month at 4℃. To a certain extent, this overcomes the problem that most LNPs currently need to be stored at ultra-low temperatures of -20℃ to -80℃. Moreover, this LNP formulation that can remain stable at refrigerated temperatures (2℃ to 8℃) also shows good therapeutic potential and good biocompatibility in terms of drug delivery.

[0171] Example 6: Treatment of HER2-positive solid tumors with 9-LNP loaded with trastuzumab-mRNA. A schematic diagram of the treatment is shown below. Figure 1 As shown

[0172] BT-474 cells were digested and collected, and after adding Matrigel, cells containing 1×10⁻⁶ cells were collected. 8 100 μL of cell suspension per cell was injected into the lower right back of each mouse to establish a subcutaneous tumor model, and the tumor was allowed to grow to 100-150 mm. 3 The size of the mice was then determined. Subsequently, the mice were randomly assigned to different treatment groups:

[0173] Control group: 100 μL of normal saline, intravenous injection (IV), n=3.

[0174] Trastuzumab: 7.5 mg / kg, intraperitoneal injection (IP), n=3.

[0175] Tucatinib (ARRY-380): 20 mg / kg, intraperitoneal injection (IP), n=3.

[0176] Trastuzumab mRNA: 2 mg / kg, intravenous injection, n=3.

[0177] HER2-siRNA: Intravenous injection (IV), n=3.

[0178] All groups were continuously treated and observed for 24 days.

[0179] Depend on Figure 39 , 40 As can be seen, compared to the direct injection of traditional monoclonal antibody drugs, the LNP delivery of trastuzumab mRNA in this invention effectively reduces off-target effects and degradation in the circulatory system, ensuring accurate transfection of the mRNA into solid tumors. Once inside tumor cells, the mRNA directs the in situ production of trastuzumab, achieving precise killing from within. This treatment strategy significantly increases the drug concentration at the tumor site while drastically reducing the drug dose in systemic circulation, thereby minimizing damage to healthy tissues.

[0180] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A cationic lipid, characterized in that, The general structural formula of the cationic lipid is: Wherein, R1 is an aminophenyl or aminoalkyl, R2 is a saturated alkyl chain or an unsaturated alkyl chain, and n is a positive integer.

2. The cationic lipid according to claim 1, characterized in that, R1 is an aminophenyl group, R2 is a saturated alkyl chain with a carbon chain length of 6-12, and n is a positive integer from 1 to 8.

3. The cationic lipid according to claim 1, characterized in that, The cationic lipid has one of the following structural formulas:

4. A lipid nanoparticle, characterized in that, The lipid nanoparticles include cationic lipids, auxiliary lipids, structural lipids, and polymeric lipids as described in any one of claims 1-3.

5. The lipid nanoparticles according to claim 4, characterized in that, In the lipid nanoparticles, the mass percentage of cationic lipids is 10-65%.

6. The lipid nanoparticles according to claim 4, characterized in that, The lipid nanoparticles have a nitrogen-to-phosphorus ratio of 2-12.

7. The lipid nanoparticles according to claim 4, characterized in that, The auxiliary lipids are selected from 1,2-distearyl-sn-glycerol-3-phosphate choline, 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine, distearyl-phosphatidyl-ethanolamine, dipalmitoylphosphatidylethanolamine, dimyristoylphosphatidylethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate choline, 1,2-dimyristoyl-sn-glycerol-3-phosphate choline, 1,2-dioleoyl-sn-glycerol-3-phosphate choline, 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline, 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline, 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline, 1,2-dipalmitoyl-sn-glycerol-3-o-4'-(N,N,N-trimethyl)homoserine, 1,2-diundecanoyl-sn-glycerol-3-phosphate choline, 1-palmitoyl- The lipid structure is selected from one or more of 2-oleoyl-sn-glycerol-3-phosphocholine, sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, and phosphatidylserine. The structural lipid is selected from one or more of cholesterol, steroids, cholesterol, fucosterol, squalene, ergosterol, stigmasterol, ergosterol, β-sitosterol, lanosterol, non-steroidal, brassosterol, cycloartenol, campesterol, and brassosterol. The polymer lipid is selected from one or more of distearylphosphatidylethanolamine polyethylene glycol, dimyristoylglycerol-3-methoxy polyethylene glycol, methoxy polyethylene glycol bistetradecylacetamide, 1,2-diacetyl-glucanol-3-methoxydimethylethylene glycol, and polyethylene glycol dicholesterol.

8. A pharmaceutical preparation, characterized in that, The pharmaceutical formulation comprises nucleic acids and the lipid nanoparticles of claim 4.

9. The pharmaceutical preparation according to claim 8, characterized in that, The nucleic acid is selected from one or more of the following: mRNA, siRNA, aiRNA, miRNA, dSRNA, shRNA, lncRNA, saRNA, rRNA, RNA, DNA, cDNA, plasmid, aptamer, tRNA, piRNA, circRNA, antisense oligonucleotide, ribozyme, PNA, and DNAzyme.

10. The use of the cationic lipids according to any one of claims 1-3 and / or the lipid nanoparticles according to any one of claims 4-7 and / or the pharmaceutical formulations according to any one of claims 8-9 in the preparation of drugs for treating solid tumors.

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