Ionizable cationic lipid analogue materials and their application as drug delivery carriers

Abstract

The present invention discloses ionizable cationic lipid analogue materials and their use as drug delivery carriers. The present invention rationally designs a series of cationic lipid analogue materials, which have mild reaction conditions, simple synthesis process, good stability, and good biocompatibility, and can achieve efficient and safe intracellular delivery of biopolymer drugs, and can also be used as delivery carriers for other types of drugs.

Description

[Technical field]

[0001] The present invention relates to the field of biomedical technology, and in particular to ionizable cationic lipid analogue materials and their application as drug delivery carriers. [Background technology]

[0002] Biopolymer drugs refer to biopolymers produced by modern biotechnology methods, derived from living organisms or synthesized in vitro, for the diagnosis, prevention or treatment of diseases. They mainly include proteins, polypeptides, antibodies, nucleic acids, etc., and play an important role in the vital activities of living organisms. Biopolymer drugs have excellent target selectivity, high efficacy, and the advantages of reliable therapeutic effects and low side effects in clinical treatment, and are becoming an important research direction for drug development. However, compared with small molecule drugs, biopolymer drugs have problems such as large size and molecular weight, negative or positive surface charge, and difficulty in passing through the cell membrane of target cells and entering the inside of the cells. In order to achieve efficient delivery of biopolymer drugs, researchers have made many efforts, and many biopolymer drug delivery carriers have been developed, such as liposomes, cationic polymers, and nanoparticles, but these carriers still suffer from the problem of poor delivery efficiency. How to improve the delivery effect of biomolecule drugs, how to maintain the stability of activity during the delivery process, how to successfully adapt to various biological barriers in the body, and how to realize and play a role in accurate delivery into cells are still the difficulties and directions of research on biomolecule drug delivery systems. Summary of the Invention [Problem to be solved by the invention]

[0003] JPEG0007644832000001.jpg38161

[0004] [In formula (I), m1 is independently selected from the group consisting of a branched alkyl group, a phenyl group, and a heteroatom-containing aromatic group; JPEG0007644832000002.jpg14167 is an alkyl group or a phenyl group, or R2 and R3 combine to form a cyclic group or a heterocyclic group, JPEG0007644832000003.jpg30165, m4 is independently a straight chain Alkylene group , ether bond-containing straight chain Alkylene group and nitrogen-containing heterocycles Alkylene group is selected from the group consisting of: In the cationic lipid analogue material of the present invention, m2 contains a tertiary amine group, thereby ensuring the pH sensitivity and tunability of the material's positive charge, and m3 contains a linear alkyl group, a linear alkenyl group, The present invention provides a method for synthesizing and producing the above-mentioned series of cationic lipid analog materials through rational design, and the cationic lipid analog materials can be bound to biopolymer drugs such as proteins and nucleic acids to form complexes, thereby achieving efficient intracellular delivery of the biopolymer drugs.

[0005] The cationic lipid analog material of the present invention, as a carrier for intracellular delivery of drugs, particularly biopolymer drugs, has high delivery efficiency, is less toxic to cells during the delivery process, and can still maintain the biological activity of the drug after it is delivered into cells. In addition, the cationic lipid analog material of the present invention can be applied as a carrier material to various proteins and nucleic acid molecules, and is a versatile and efficient intracellular delivery carrier for biopolymer drugs. In the above formula (I), m1 is an alkyl group, a phenyl group or a phenyl group substituted with a substituent α. The resulting cationic lipid analog materials have high efficiency for intracellular delivery of proteins. JPEG0007644832000006.jpg154170Further, in the above formula (I), m4 is a linear alkyl group having 6 carbon atoms. Alkylene group , straight chain containing ether bonds with 4 to 8 carbon atoms Alkylene group or containing a nitrogen-containing heterocycle Alkylene group and JPEG0007644832000007.jpg27170 Analogue materials are highly efficient at delivering proteins intracellularly. Additionally, the cationic lipid analog material has any one of the following 72 structures: JPEG0007644832000008.jpg212131JPEG0007644832000009.jpg186132JPEG0007644832000010.j pg185132JPEG0007644832000011.jpg235132JPEG0007644832000012.jpg226132JPEG00076448320 00013.jpg168132JPEG0007644832000014.jpg186131JPEG0007644832000015.jpg202131JPEG000 7644832000016.jpg203129JPEG0007644832000017.jpg198131JPEG0007644832000018.jpg196132

[0006] The inventors have found through testing that the 72 kinds of small molecular cationic lipid analogue materials screened above can be co-assembled with protein model drugs to form small and stable nanocomplexes, which can achieve intracellular delivery of various positive and negative proteins, and can maintain the biological activity of the proteins delivered into cells and achieve therapeutic effects.In addition, these 72 kinds of small molecular cationic lipid analogue materials can also be combined with various nucleic acid molecules to form nanoparticles, which can achieve efficient intracellular delivery of plasmid DNA, mRNA and siRNA. The present invention further provides a method for producing the above cationic lipid analog material. Specifically, the method includes adding an aldehyde compound and an amine compound to an organic solution, reacting them for 10 to 120 minutes, and then reacting them with a carboxylic acid compound. IsocyanideThe compounds are added in order and reacted at 4 to 60° C. for 6 to 72 hours. After the reaction is completed, the product is separated and purified by column chromatography to obtain the cationic lipid analog material.

[0007] In the present invention, an aldehyde compound, an amine compound, a carboxylic acid compound, and Isocyanide The compound is used as raw material to synthesize a low molecular weight cationic lipid analogue material by Ugi reaction. The reaction conditions of the cationic lipid analogue material of the present invention are mild, the synthesis process is simple, and the stability is good. The synthesized low molecular weight cationic lipid analogue material has low toxicity, and can efficiently deliver a variety of drugs into cells. Furthermore, in the method for producing the cationic lipid analogue material, separation is carried out by column chromatography using a mixture of methanol and dichloromethane as the mobile phase. Furthermore, in the method for producing the cationic lipid analog material, the aldehyde compound, the amine compound, the carboxylic acid compound, and Isocyanide The molar ratio of the compounds is 0.1-1:0.1-1:0.1-1:0.1-1, and further, the molar ratio is 1:1:1:0.5. Furthermore, in the method for producing a cationic lipid analog material, the aldehyde compound is any one selected from the group consisting of the following compounds A1 to A3: JPEG0007644832000019.jpg3835Furthermore, the aldehyde compound is compound A1,

[0008] The amine compound is any one selected from the group consisting of the following compounds R1 to R11: JPEG0007644832000020.jpg14337 The carboxylic acid compound is any one selected from the group consisting of the following compounds CHS, C18-1, C18-2, and C8 to C20, JPEG0007644832000021.jpg16475 IsocyanideThe compound is any one selected from the group consisting of the following compounds I1, I2-1, I2, I2-3, and I3. JPEG0007644832000022.jpg3955

[0009] The present invention further provides the use of the above cationic lipid analogue material as a drug carrier. The low molecular weight cationic lipid analogue material of the present invention can form a stable complex advantageous for cellular uptake by interacting with various proteins, and the complex can effectively utilize pathways such as cell fusion and / or endocytosis to cross the cell membrane, avoid and / or escape from endosomes, and deliver proteins into cells, while still maintaining the biological activity of the protein delivered into the cells, and the cationic lipid analogue material does not cause toxicity to cells during the delivery process, and has excellent biocompatibility. In addition, the cationic lipid analogue material of the present invention can efficiently bind to nucleic acid molecules such as plasmid DNA, mRNA, and siRNA, and can deliver different plasmid DNAs to various types of cells, all of which show high transfection efficiency, which is equal to or higher than that of currently commercially available transfection reagents. As a result, the cationic lipid analogue material designed in the present invention can be used as an intracellular delivery carrier for drugs, particularly biopolymers, and can provide an effective tool for the therapeutic application of biopolymer drugs.

[0010] The present invention further provides a drug delivery system comprising a carrier employing the above-mentioned cationic lipid analogue material and a drug encapsulated or loaded in the carrier. The drug delivery system is not particularly limited in the type of drug, and includes one or both of biological drugs and chemical drugs, but is not limited thereto. Specifically, the biological drugs include one or more of nucleic acids, polypeptides, proteins, and vaccines, but are not limited thereto. The chemical drugs include one or more of antitumor small molecule drugs, fluorescein, and lymphatic tracers, but are not limited thereto.

[0011] To further improve the targeting and delivery effect of the drug delivery system, the carrier can be directly conjugated or chemically conjugated with a targeting ligand, specifically, the targeting ligand can include, but is not limited to, an oligonucleotide-based adaptor, an antibody molecule, an antibody fragment, a naturally occurring receptor surface conjugate, or a polypeptide, etc. The targeting ligand can be manufactured or synthesized by one or more chemical modification methods, where the chemical modification methods include, but are not limited to, fluoro modification, methoxy group modification, amino group modification, locked nucleic acid, glycosylation, amino group modification, or phosphorylation.

[0012] Within the scope of the present invention, the drug delivery system may further contain any pharmacologically acceptable additive to prepare dosage forms such as solution-type liquid preparations, polymer solutions, emulsions, suspensions, dispersions, granules, tablets and capsules, and the additives include, but are not limited to, one or more of excipients, binders, diluents and disintegrants.

[0013] As used herein, the term "delivery" or "intracellular delivery" means that a drug enters the cell from the outside to the inside of the cell and localizes it in the cytoplasm or in an organelle of the cell.

[0014] As used herein, the term "complex" of a cationic lipid analog material with a biopolymer drug such as a protein or nucleic acid, or a "complex" consisting of a cationic lipid analog material and a biopolymer, refers to an interaction between a cationic lipid analog material and a biopolymer drug such as a protein or nucleic acid, which is sufficiently stable to bind the biopolymer drug and the cationic lipid analog material and deliver them into cells.

[0015] As used herein, the term "nanoparticles" refers to particles having a diameter of less than 1 μm. Compared with the prior art, the beneficial effects of the present invention are as follows:

[0016] In the present invention, an aldehyde compound, an amine compound, a carboxylic acid compound, and Isocyanide The compound is used as raw material to synthesize low molecular weight cationic lipid analogue material by Ugi reaction. The reaction conditions of the cationic lipid analogue material of the present invention are mild, the synthesis process is simple, and the stability is good. The synthesized low molecular weight cationic lipid analogue material has good biocompatibility, and can achieve safe and efficient intracellular delivery of various drugs. [Brief description of the drawings]

[0017] [Figure 1] 1A and 1H NMR spectra (b) of the cationic lipid analog material I2-1R2C18A1. [Diagram 2] 1A and 1H NMR spectra (b) of the cationic lipid analog material I2R2C18A1. [Diagram 3] 1A and 1H NMR spectra (b) of the cationic lipid analog material I2-3R2C18A1. [Figure 4]Figure 1 shows the mean fluorescence intensity of fluorescently labeled proteins in HeLa cells after delivery of BSA-FITC with different cationic lipid analog materials. The doses of I1R1C12A1, I2R1C14A1 and I2R3C16A1 were 1 μg / well, and the doses of I1R2C14A1, I1R2C16A1, I1R2C18A1, I1R3C18A1, I1R11C18A1, I2R1C12A1, I2R1C20A1, I2R2C16A1, I2R3C18A1, I2R11C14A1, I2R11C16A1 and I2R11C18A1 were 2 μg / well during the experiment. The doses of I1R3C14A1, I1R3C16A1, I1R3C20A1, I1R11C14A1, I1R11C16A1, I1R11C20A1, I2R2C18A1, I2R2C20A1, I2R3C20A1 and I2R11C20A1 were 4 μg / well, and the doses of I1R1C14A1, I1R1C16A1, I1R1C18A1, I1R1C20A1, I1R2C12A1, I1R2C20A1, I1R3C12A1, I1R5 The dose of C12A1, I1R5C14A1, I1R5C16A1, I1R5C18A1, I1R5C20A1, I1R11C12A1, I2R1C16A1, I2R1C18A1, I2R2C12A1, I2R2C14A1, I2R3C12A1, I2R3C14A1, I2R5C12A1, I2R5C14A1, I2R5C16A1, I2R5C18A1, I2R5C20A1 and I2R11C12A1 is 8 μg / well. The dose of BSA-FITC is 2 μg / well. [Diagram 5] Fluorescently labeled protein positive rates of HeLa cells after delivery of BSA-FITC with different cationic lipid analog materials, where the experimental conditions are consistent with those described in FIG. [Figure 6] Average fluorescence intensity of fluorescently labeled proteins in HeLa cells after delivery of BSA-FITC with cationic lipid analog materials with different alkyl chain lengths, where the dose of cationic lipid analog is 4 μg / well and the dose of BSA-FITC is 2 μg / well. [Figure 7]BSA-FITC intracellular delivery efficiency corresponding to I2-1R2C18A1, I2R2C18A1 and I2-3R2C18A1 (a) and particle size distribution of the corresponding complexes (b). [Figure 8] Laser confocal results of I2-1R2C18A1 delivery of BSA-FITC to different cell types. [Figure 9] 1 shows the cytotoxicity results of I2R2C16A1, I2R2C17A1, I2R2C18A1, I2R2C19A1, I2R2C20A1 and their respective conjugates with BSA-FITC. [Figure 10] 2 shows the intracellular delivery efficiency of I2R2C18A1, I2-1R2C18A1 and the positive control PULSin® relative to phycoerythrin, a negatively charged protein. [Figure 11] Intracellular delivery efficiency of I2-1R2C18A1 for superoxide dismutase, a negatively charged protein. [Figure 12] Intracellular delivery efficiency of I2-1R2C18A1 for ovalbumin, a negatively charged protein. [Figure 13] Intracellular delivery efficiency of I2-1R2C18A1 for green fluorescent protein, a negatively charged protein. [Figure 14] Intracellular delivery efficiency of I2-1R2C18A1 for cytochrome C, a positively charged protein. [Figure 15] Intracellular delivery efficiency of I2-1R2C18A1 for lysozyme, a positively charged protein. [Figure 16] β-galactosidase activity results in HeLa cells after delivery of protein β-galactosidase, where the dose of cationic lipid analogue is 4 μg / well and the dose of β-galactosidase is 4 μg / well. [Figure 17] Figure 1 shows the cellular activity results of HeLa cells after delivery of the protein saporin with I2-1R2C18A1, where the dose of the cationic lipid analogue is 3 μg / well. [Figure 18]The positive rate results when GFP expression plasmid DNA was transfected with different cationic lipid analog materials, where the dose of I2R11C14A1 and I2R11C18-2A1 was 0.5 μg / well, the dose of I1R2C14A1, I2R1C16A1, I2R1C18A1, I2R1C18-1A1, I2R1C18-2A1, I2R2C18-2A1, I2R3C14A1, I2R3C16A1 and I2R3C18-2A1 was 1 μg / well, and the dose of I1R2C16A1, I1R2C18-1A1, I1 The doses of R2C18-2A1, I1R3C14A1, I1R3C16A1, I1R3C18-2A1, I1R11C14A1, I1R11C16A1, I1R11C18A1, I1R11C18-1A1, I1R11C18-2A1, I2R1C14A1, I2R2C14A1, I2R2C16A1, I2R2C18-1A1, I2R3C18A1, I2R3C18-1A1 and I2R11C16A1 were 2 μg / mL. and the dose of I1R1C16A1, I1R2C18A1, I1R3C12A1, I1R3C18A1, I1R3C18-1A1, I1R11C12A1, I2R2C18A1, I2R11C12A1, I2R11C18A1 and I2R11C18-1A1 was 4 μg / well, and the dose of I1R1C12A1, I1R1C14A1, I1R1C18A1, I1R1C18-1A1, I1R1C18-2A1 , I1R2C12A1, I1R5C12A1, I1R5C14A1, I1R5C16A1, I1R5C18A1, I1R5C18-1A1, I1R5C18-2A1, I2R1C12A1, I2R2C12A1, I2R3C12A1, I2R5C12A1, I2R5C14A1, I2R5C16A1, I2R5C18A1, I2R5C18-1A1 and I2R5C18-2A1 at a dose of 8 μg / well. [Figure 19] 1 shows the results of detecting the amount of luciferase expression in HeLa cells after transfection of luciferase expression plasmid DNA with different cationic lipid analogue materials. [Figure 20] Laser confocal images of GFP expression plasmid DNA transfected into different types of cells with different amounts of I2R3C18-1A1 added. [Figure 21] The positive rate results when eGFP-mRNA was transfected with different cationic lipid analog materials. Here, I1R2C14A1, I1R2C18-1A1, I1R2C18-2A1, I1R11C14A1, I2R2C14A1, I2R2C18-1A1, I2R2C18-2A1, I2R3C16A1, I2R3C18A1, I2R3C18-1A1, I2R3C18-2A1, I2R11C14A1, I2R11C16A1, I2R11C18-1A1 and I2R11C18 The dose of -2A1 was 1 μg / well, and the following combinations were used: I1R1C12A1, I1R1C14A1, I1R1C16A1, I1R1C18A1, I1R1C18-1A1, I1R1C18-2A1, I1R2C12A1, I1R2C16A1, I1R2C18A1, I1R3C12A1, I1R3C14A1, I1R3C16A1, I1R3C18A1, I1R3C18-1A1, I1R3C18 -2A1, I1R5C12A1, I1R5C14A1, I1R5C16A1, I1R5C18A1, I1R5C18-1A1, I1R5C18-2A1, I1R11C12A1, I1R11C 16A1, I1R11C18A1, I1R11C18-1A1, I1R11C18-2A1, I2R1C12A1, I2R1C14A1, I2R1C16A1, I2R1C18A1, I2R1 The dose of C18-1A1, I2R1C18-2A1, I2R2C12A1, I2R2C16A1, I2R2C18A1, I2R3C12A1, I2R3C14A1, I2R5C12A1, I2R5C14A1, I2R5C16A1, I2R5C18A1, I2R5C18-1A1, I2R5C18-2A1, I2R11C12A1 and I2R11C18A1 is 2 μg / well. [Figure 22] 21 shows the mean fluorescence intensity upon transfection of eGFP-mRNA with different cationic lipid analogue materials, where the experimental conditions are consistent with those in FIG. [Figure 23]These are the results of laser confocal imaging when eGFP-mRNA was transfected into DC2.4 cells using I2R2C18-2A1, I2R3C18-2A1, or I2R11C18-2A1. The commercially available transfection reagent Lipofectamine 2000 was used as a positive control. [Figure 24] This shows the results of gene silencing when siRNA was transfected with different amounts of I2R2C18-1A1, I2R3C18-1A1, I2R2C18-2A1, and I2R3C18-2A1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] In order to better illustrate the objectives, technical solutions and advantages of the present invention, the present invention will be described below with reference to specific examples. Those skilled in the art should understand that the specific examples described herein are only for illustrating the present invention, and are not intended to limit the present invention.

[0019] In the examples, the experimental methods employed are conventional unless otherwise stated, and the materials, reagents etc. used are commercially available unless otherwise stated. Example 1 Synthesis and Identification of Cationic Lipid Analogue Materials The synthetic route for the cationic lipid analogue materials of the present invention is as follows. JPEG0007644832000023.jpg14146JPEG0007644832000024.jpg55165 JPEG0007644832000025.jpg144147. The method for producing the cationic lipid analog material of this embodiment is specifically as follows: 1 mmol of isobutyraldehyde and 1 mmol of an amine compound are each placed in 0.5 mL of methanol solution, reacted for 60 minutes, and then 1 mmol of a carboxylic acid compound and 0.5 mmol of a carboxylic acid compound are added to the methanol solution. IsocyanideThe compounds are added in order and reacted at 40° C. for 12 hours. After the reaction is completed, the product is separated and purified by column chromatography, in which a mixture of methanol and dichloromethane is used as the mobile phase. The structures of the starting materials and cationic lipid analog materials synthesized in this example are shown in Table 1. JPEG0007644832000026.jpg217164JPEG0007644832000027.jpg236164JPEG0007644832000028.jpg199 164JPEG0007644832000029.jpg200164JPEG0007644832000030.jpg200164JPEG0007644832000031.jpg2 42164JPEG0007644832000032.jpg200164JPEG0007644832000033.jpg242164JPEG0007644832000034.j pg187164JPEG0007644832000035.jpg242164JPEG0007644832000036.jpg242164JPEG0007644832000037 .jpg200164JPEG0007644832000038.jpg242164JPEG0007644832000039.jpg242164JPEG0007644832000 040.jpg212164JPEG0007644832000041.jpg242164JPEG0007644832000042.jpg206164JPEG00076448320 00043.jpg242164JPEG0007644832000044.jpg242164JPEG0007644832000045.jpg206164JPEG0007644832000046.jpg121164The cationic lipid analogs I2-1R2C18A1, I2R2C18A1 and I2-3R2C18A1 were selected as representative materials and their structures were identified. Here, the mass spectrum and 1 The H NMR spectrum is shown in Figure 1, and the mass spectrum of I2R2C18A1 and 1 The H NMR spectrum is shown in FIG. 2, and the mass spectrum of I2-3R2C18A1 and1 The H NMR spectrum is shown in Figure 3. 1 The 1 H NMR and mass spectral results were consistent with the predicted structures of the cationic lipid analogue materials.

[0020] Example 2 In this example, the application effect of cationic lipid analogue materials on intracellular delivery of proteins was investigated using fluorescein isothiocyanate-labeled bovine serum albumin (BSA-FITC) as a protein model. The specific experimental method is as follows. HeLa cells were inoculated into a 24-well plate in advance and cultured in a cell culture incubator for 12 hours. Then, different cationic lipid analog materials (0.25-8 μg / well) were mixed with BSA-FITC (2 μg / well) in 50 μL of HEPES buffer, respectively, and diluted with 450 μL of serum-free DMEM medium to obtain protein / cationic lipid analog complex solutions. The medium of HeLa cells was removed, washed once with PBS, and then the protein / cationic lipid analog complex solution was added. After culturing for 4 hours, the intracellular fluorescence intensity and positive cell rate were analyzed by flow cytometry. In this experiment, the commercially available protein delivery reagent PULSin (registered trademark) was used as a positive control. From the results of Figures 4 and 5, it can be seen that all of the cationic lipid analog materials of the present invention are effective for intracellular delivery of BSA-FITC, and in particular, Isocyanide It was found that when the compound was I2 and the amine compound was R2, R3 or R11, most of the synthesized cationic lipid analog materials had significantly better delivery efficiency than the commercially available protein delivery reagent PULSin®. In this experiment, the average fluorescence intensity of cellular proteins was compared for cationic lipid analogue materials with different alkyl chain lengths, using I2R2C14A1, I2R2C15A1, I2R2C16A1, I2R2C17A1, I2R2C18A1, I2R2C19A1, and I2R2C20A1 as representatives. As can be seen from the results in Figure 6, in the present invention, the hydrophobicity of the cationic lipid analogue material can be adjusted by increasing the alkyl chain length of the carboxylic acid compound, and the intracellular delivery efficiency of protein by the cationic lipid analogue material is improved with the increase in alkyl chain length, and reaches a plateau value when the alkyl chain of the carboxylic acid compound has 18 carbon atoms. In this experiment, the particle size of the protein / cationic lipid analogue complex was measured using I2-1R2C18A1, I2R2C18A1 and I2-3R2C18A1 as representatives. As can be seen from the results in Figure 7, I2-1R2C18A1, I2R2C18A1 and I2-3R2C18A1 all have excellent intracellular protein delivery efficiency (the dose of the cationic lipid analogue is 4 μg / well, and the dose of BSA-FITC is 2 μg / well.), and they form a complex with small size and uniform distribution with BSA protein, and the particle size of the complex is about 500 nm.

[0021] Example 3. Protein delivery effect of I2-1R2C18A1 in different types of cells In this experiment, I2-1R2C18A1 was used as a representative cationic lipid analogue material to investigate the protein delivery effects of different types of cationic lipid analogue materials in cells. In this experiment, the method of Example 2 was used to prepare a BSA-FITC / I2-1R2C18A1 complex solution, in which I2-1R2C18A1 was 4μg / well and BSA-FITC was 4μg / well. The BSA-FITC / I2-1R2C18A1 complex solution was added to human kidney epithelial cells (HEK-293T), human pancreatic cancer cells (BxPC3), mouse macrophages (RAW 264.7), mouse dendritic cells (DC 2.4), human umbilical vein endothelial cells (HUVEC), and mouse mesenchymal stem cells (MSC), respectively, and the solution was cultured for 4 hours. Then, the FITC fluorescent signal in the cells was observed using a laser confocal microscope. The results in Figure 8 show that I2-1R2C18A1 can transfect BSA-FITC into human renal epithelial cells (HEK-293T), human pancreatic cancer cells (BxPC3), immune cells (mouse macrophages (RAW 264.7) and mouse dendritic cells (DC 2.4), endothelial cells (human umbilical vein endothelial cells (HUVEC) and endothelial cells (mouse mesenchymal stem cells (MSC)). As can be seen, I2-1R2C18A1 showed good protein delivery effects in different types of cells.

[0022] Example 4 Cytotoxicity Testing of Cationic Lipid Analogue Materials and Their Protein Conjugates In this experiment, I2R2C16A1, I2R2C17A1, I2R2C18A1, I2R2C19A1, and I2R2C20A1, which have high protein delivery efficiency, were selected as representative cationic lipid analog materials, and the toxicity of protein / cationic lipid analogs to HeLa cells was detected by MTT experiments. The specific experimental method was as follows: HeLa cells were spread on a 96-well plate and cultured in a cell incubator for 12 hours, after which the cell medium was removed and replaced with 1 μg / well of cationic lipid analog materials or BSA-FITC / cationic lipid analog complexes (the mass ratio of cationic lipid analog materials to BSA-FITC is 2:1), and cultured for 4 hours, after which the materials were removed by washing, and the medium was replaced with DMEM medium and cultured for 20 hours, and finally the cell viability was detected by MTT method. The results in FIG. 9 demonstrate that the cationic lipid analog material of the present invention and its protein complex have good biocompatibility.

[0023] Example 5. Intracellular delivery effect of cationic lipid analogue materials on different proteins In this experiment, the intracellular delivery effect of cationic lipid analogue materials was examined for phycoerythrin (R-PE), superoxide dismutase, ovalbumin, green fluorescent protein, cytochrome C, and lysozyme. In this experiment, a protein / cationic lipid analogue complex solution was prepared with reference to the method in Example 2, and its delivery effect in HeLa cells was examined. Here, the delivery effects of I2R2C18A1, I2-1R2C18A1 and the positive control PULSin® on phycoerythrin, a negatively charged protein, are shown in FIG. 10, the delivery effect of I2-1R2C18A1 on superoxide dismutase, a negatively charged protein, is shown in FIG. 11, the delivery effect of I2-1R2C18A1 on ovalbumin, a negatively charged protein, is shown in FIG. 12, the delivery effect of I2-1R2C18A1 on green fluorescent protein, a negatively charged protein, is shown in FIG. 13, the delivery effect of I2-1R2C18A1 on cytochrome C, a positively charged protein, is shown in FIG. 14, and the delivery effect of I2-1R2C18A1 on lysozyme, a positively charged protein, is shown in FIG. 15. The above results demonstrate that the cationic lipid analog material of the present invention can efficiently deliver proteins with different molecular weights and charge characteristics, such as bovine serum albumin, phycoerythrin, superoxide dismutase, ovalbumin, green fluorescent protein, cytochrome C, and lysozyme, into cells, demonstrating that the cationic lipid analog material of the present invention is versatile in terms of intracellular delivery of protein drugs.

[0024] Example 6 β-galactosidase (β-Gal) was delivered by I2-1R2C18A1, and its intracellular activity was detected. After HeLa cells were cultured with β-Gal, β-Gal / PULSin, or β-Gal / I2-1R2C18A for 4 hours, the cells were washed with PBS and the activity of β-galactosidase in the cells was detected using a β-Gal in situ assay kit according to the manufacturer's instructions. In this experiment, the commercially available protein transfection reagent PULSin® was used as a positive control. Maintaining the biological activity of a compound is an important principle of the delivery technology of biologically active molecules. As can be seen from the results in Figure 16, I2-1R2C18A1 can efficiently deliver β-Gal into cells and maintain its biological activity, and its effect is significantly superior to that of the commercially available protein delivery reagent PULSin®.

[0025] Example 7 Saporin was delivered to HeLa cells using I2-1R2C18A1 and the cellular activity was detected HeLa cells were incubated with saporin or saporin / I2-1R2C18A1 for 4 hours, then the medium was replaced with complete medium and cultured for another 20 hours, after which cell activity was detected by the MTT assay. The results in Figure 17 show that saporin itself showed little toxicity to cells, but I2-1R2C18A1 has high protein delivery efficiency and can effectively deliver saporin into cells, and that the saporin delivered into cells has a therapeutic effect and can effectively kill tumor cells.

[0026] Example 8 Transfection experiment of plasmid DNA for expressing green fluorescent protein (GFP) In this experiment, a plasmid DNA for expressing green fluorescent protein (GFP) was used as a reporter gene to detect the gene transfection efficiency of cationic lipid analogue materials in HeLa cells. The specific method is as follows. HeLa cells were seeded in a 24-well plate and cultured in a cell incubator for 12 hours. Different cationic lipid analogue materials (0.25-8 μg / well) were mixed with plasmid DNA (0.5 μg / well) for green fluorescent protein expression in 40 μL of sodium acetate buffer (25 mM, pH 5.2), left to stand for 10 minutes, and then further diluted with 460 μL of Opti-MEM medium to obtain a cationic lipid analogue complex particle solution loaded with plasmid DNA. After removing the HeLa cell medium and washing once with PBS, the complexed particle solution was added and cultured for 24 hours, and the transfection efficiency of the plasmid DNA in the cells was analyzed by flow cytometry. As a positive control, a commercially available gene transfection reagent Lipofectamine 2000 was used. From the results of FIG. 18, it can be seen that the cationic lipid analogue material of the present invention can transfect a plasmid DNA expressing GFP into HeLa cells, realizing efficient gene transfection, and in particular, I1R2C14A1, I1R2C18-2A1, I1R11C14A1, I2R1C14A1, I2R1C16A1, I2R1C18-1A1, I2R1C18-2A1, I2 It was found that R2C14A1, I2R2C16A1, I2R2C18-1A1, I2R2C18-2A1, I2R3C16A1, I2R3C18A1, I2R3C18-1A1, I2R3C18-2A1, I2R11C14A1, I2R11C16A1, I2R11C18A1, I2R11C18-1A1, and I2R11C18-2A1 may have transfection efficiency equivalent to or higher than that of the commercially available transfection reagent Lipofectamine 2000.

[0027] Example 9 Transfection experiment of plasmid DNA for expressing luciferase (luminescence) In this example, the luciferase expression plasmid DNA was used as a reporter gene to detect the gene transfection efficiency of cationic lipid analogue materials in HeLa cells, and the specific operation method is as follows: HeLa cells were seeded into a 96-well plate and cultured in a cell incubator for 12 hours. Different cationic lipid analogue materials were mixed with luciferase expression plasmid DNA (0.5 μg) in 40 μL of sodium acetate buffer (25 mM, pH 5.2) and left to stand for 10 minutes, and then further diluted with 460 μL of Opti-MEM medium to obtain a cationic lipid analogue complex particle solution loaded with plasmid DNA. The HeLa cell medium was removed, washed once with PBS, and then 125 μL of the complexed particle solution was added. After 24 hours of culture, the culture solution was removed and fully dissolved, and 50 μL / well of substrate was added, and the expression amount of luciferase was detected using a multi-function plate reader. The results in Figure 19 demonstrate that I2R3C18A1, I2R1C18-1A1, I1R11C14A1, I2R11C18-2A1, I1R2C14A1, I2R1C14A1, I2R1C16A1, I2R3C18-2A1, I2R2C18-2A1, I2R2C16A1, I2R3C16A1, I2R2C18-1A1, and I2R3C18-1A1 may have transfection efficiency equivalent to or greater than that of the commercially available transfection reagent Lipofectamine 2000.

[0028] Example 10 Transfection efficiency of plasmid DNA for GFP expression by I2R3C18-1A1 into different types of cells In this experiment, I2R3C18-1A1 was used as a representative cationic lipid analog material, and GFP expression plasmid DNA was used as a reporter gene to study the transfection effect of different amounts of cationic lipid analog material (0.5-3 μg / well) on plasmid DNA (0.5 μg / well) in different types of cells. The commercially available gene transfection reagent Lipofectamine 2000 was used as a positive control. In this experiment, the I2R3C18-1A1 particle solution loaded with GFP expression plasmid DNA was prepared with reference to the method in Example 8, and the I2R3C18-1A1 particle solution complex solution loaded with GFP expression plasmid DNA was added to DC2.4, RAW 264.7, A549, BxPC3 and HeLa cells, respectively, and cultured for 24 hours. After that, the transfection efficiency of the plasmid DNA in the cells was observed using a laser confocal microscope. The results in FIG. 20 demonstrated that the I2R3C18-1A1 material was capable of transfecting GFP-expressing plasmid DNA into tumor cells and immune cells.

[0029] Example 11 Transfection experiment of mRNA for enhanced green fluorescent protein (eGFP) expression In this experiment, eGFP-mRNA was used as the mRNA model to detect the mRNA transfection efficiency of cationic lipid analogue materials in DC2.4 cells. The specific operation method is as follows: DC 2.4 cells were seeded on a 48-well plate and cultured in a cell incubator for 12 hours. Different cationic lipid analog materials (0.25-2 μg / well) were mixed with eGFP expression mRNA (0.2 μg / well) in 20 μL of sodium acetate buffer (25 mM, pH 5.2), respectively, and left to stand for 10 minutes, after which they were further diluted with 230 μL of Opti-MEM medium to obtain composite particles loaded with mRNA. The HeLa cell medium was removed, washed once with PBS, and then the composite particle solution was added. After culturing for 24 hours, the intracellular mRNA transfection efficiency was observed by flow cytometry and laser confocal microscopy. As a positive control, a commercially available gene transfection reagent Lipofectamine 2000 was used. From the results of Figures 21 to 23, it can be seen that the cationic lipid analogue material of the present invention can transfect eGFP expression mRNA into DC2.4 cells, and in particular, I1R2C14A1, I1R2C16A1, I1R2C18A1, I1R2C18-1A1, I1R2C18-2A1, I1R11C14A1, I1R11C16A1, I1R11C18A1, I2R1C14A1, I2R1C16A1, I2R1C18-1A1, I2R2C14A1, I2R2C16A1, I2 ...2C16A1, I2R2C18-2A1, I1R11C14A1, I1R11C16A1, I2R1C18-2A1, I1R11C14A1, I1R11C16A1, I2R1C18-2A1, I1R11C14A1, I1R11C16A1, I2R1C18-2A1, I1R11C14A1, I1R11C16A1, I2R1C18-2A1, I1R11C14A1, I1R It was found that 18A1, I2R2C18-1A1, I2R2C18-2A1, I2R3C14A1, I2R3C16A1, I2R3C18A1, I2R3C18-1A1, I2R3C18-2A1, I2R11C14A1, I2R11C16A1, I2R11C18A1, I2R11C18-1A1, and I2R11C18-2A1 can have mRNA transfection efficiency equivalent to or higher than that of the commercially available transfection reagent Lipofectamine 2000.

[0030] Example 12 Short interfering nucleic acid (siRNA) transfection experiments In this experiment, I2R2C18-1A1, I2R3C18-1A1, I2R2C18-2A1, and I2R3C18-2A1 were selected as representative cationic lipid analog materials, A549 cells (A549-Luc) stably expressing luciferase reporter gene were selected as cell models, and the siRNA sequence specifically targeting the luciferase reporter gene was used as the siRNA model to study the effect of siRNA transfection and delivery of cationic lipid analog materials, and the specific operation method is as follows: A549 cells expressing luciferase (A549-Luc) were seeded in a 96-well plate and cultured in a cell culture incubator for 12 hours. Different materials (0.5-3 μg / well) were mixed with siRNA in 40 μL of sodium acetate buffer (25 mM, pH 5.2), respectively, and left to stand for 10 minutes, then diluted with 460 μL of Opti-MEM medium to obtain composite particles loaded with siRNA (final concentration of siRNA is 100 nM). The medium of A549-Luc cells was removed, washed once with PBS, and 125 μL of composite particle solution was added, and cultured for 48 hours. After removing the culture medium, the cells were thoroughly dissolved, and 50 μL / well of substrate was added, and the expression level of luciferase was detected using a multi-function plate reader. The results in Figure 24 show that I2R2C18-1A1, I2R3C18-1A1, I2R2C18-2A1, and I2R3C18-2A1 can transfect siRNA into cells to specifically silence the expression of luciferase reporter gene, and the gene silencing efficiency improves with increasing amounts of cationic lipid analogue materials added. JPEG0007644832000047.jpg98161

Claims

1. An ionizable cationic lipid analog material having a structure represented by formula (I): [In formula (I), m １ are independently selected from the group consisting of a straight chain alkyl group, a branched alkyl group, a phenyl group, and a heteroatom-containing aromatic group; R ３ is an alkyl group or a phenyl group, or R ２ and R ３ are linked to form a cyclic group or m ３ are independently a straight chain alkyl group, a straight chain alkenyl group or m ４ are independently selected from the group consisting of a straight-chain alkylene group, an ether bond-containing straight-chain alkylene group, or an alkylene group containing a nitrogen-containing heterocycle.

2. m １ 2. The cationic lipid analog material of claim 1, wherein: is an alkyl group, a phenyl group, or a heteroatom-containing aromatic group substituted with a substituent α, said substituent comprising a methyl group.

3.

4.

5. m ３ is a linear alkyl group having 7 to 19 carbon atoms, a linear alkenyl group having 17 carbon atoms, or

6.

7. m ４ is a straight-chain alkylene group having 6 carbon atoms, a straight-chain alkylene group containing an ether bond having 4 to 8 carbon atoms, or an alkylene group containing a nitrogen-containing heterocycle. The cationic lipid analog material according to claim 1,

8.

9. 2. The cationic lipid analog material of claim 1, characterized in that it has any of the following 72 structures:

10. A method for producing a cationic lipid analogue material according to any one of claims 1 to 9, comprising: adding an aldehyde compound and an amine compound to an organic solution, reacting for 10 to 120 min, and then adding a carboxylic acid compound and an isocyanide compound in sequence, reacting at 4 to 60°C for 6 to 72 h, and after the reaction is completed, isolating and purifying the product by column chromatography to obtain the cationic lipid analogue material.

11. The method for producing a cationic lipid analogue material according to claim 10, characterized in that the molar ratio of the aldehyde compound, the amine compound, the carboxylic acid compound and the isocyanide compound is 1:1:1:0.1-1.

12. The method for producing a cationic lipid analog material described in claim 11, characterized in that the molar ratio of the aldehyde compound, the amine compound, the carboxylic acid compound and the isocyanide compound is 1:1:1:0.

5.

13. The aldehyde compound is any one selected from the group consisting of the following compounds A1 to A3: The amine compound is any one selected from the group consisting of the following compounds R1 to R11: The carboxylic acid compound is any one selected from the group consisting of the following compounds CHS, C18-1, C18-2, and C8 to C20, The isocyanide compound is any one selected from the group consisting of the following compounds I1, I2-1, I2, I2-3, and I3: A method for producing the cationic lipid analogue material according to claim 10.

14. Use of the cationic lipid analogue material according to any one of claims 1 to 9 as a drug carrier.

15. A drug delivery system comprising a carrier which is a cationic lipid analogue material according to any one of claims 1 to 9, and a drug encapsulated or loaded in the carrier.

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