Esterase-responsive cationic lipid molecules for gene delivery

Abstract

The application discloses an esterase response cationic lipid molecule for gene delivery, wherein the cationic lipid molecule is designed with a large number of positive charges, can stably wrap nucleic acid drugs, and can remove the positive charges by the action of esterase after entering cells, so that charge reversal occurs, the system is neutral or negative, and the nucleic acid drugs can be quickly released for transfection. The nanoparticles formed by the cationic lipid have higher transfection efficiency than commercial liposomes, lower toxicity than commercial gene carriers, and have good application prospect.

Description

Technical Field

[0001] This invention relates to the field of nanobiomaterials production technology, and in particular to an esterase-responsive cationic lipid molecule for gene delivery. Background Technology

[0002] The working principle of gene vaccines is as follows: after delivering the antigen gene to the cytoplasm or nucleus of the host cell, the host cell regards it as an endogenous gene and uses the host cell's transcription and translation mechanisms to produce related antigens, triggering an adaptive immune response (IM Alfagih, B. Aldosari, B. AlQuadeib, et al., Nanoparticles as Adjuvants and Nanodelivery Systems for mRNA-Based Vaccines. Pharmaceutics, 2020.13(1)). Compared with traditional vaccines such as attenuated vaccines, inactivated pathogens, and subunit vaccines, they have the advantages of no metabolic toxicity, simultaneous induction of humoral and cellular immunity, and short development cycle. Among the many delivery systems, non-viral vectors such as liposomes or lipid nanoparticles prepared using lipid or lipid-like materials dominate. Lipid nanoparticles used for gene delivery typically contain four components: amine-containing lipids or lipid-like molecules, phospholipid molecules, cholesterol, and lipid-anchored polyethylene glycol (JBMiller and DJSiegwart, Design of synthetic materials for intracellular delivery of RNAs: From siRNA-mediated gene silencing to CRISPR / Cas gene editing. Nano Research, 2018.11(10):5310-5337.). Amine-containing lipids usually contain ionizable amino structures such as tertiary amines, which are protonated at pH below the amino pKa and bind to negatively charged DNA or RNA through electrostatic interactions. Phospholipid molecules participate in membrane formation, cholesterol promotes membrane fusion and improves the stability of nanoparticles, and lipid-anchored polyethylene glycol can prolong the half-life of nanoparticles (TMAllen and PRCullis, Liposomal drug delivery systems: from concept to clinical applications. Adv Drug Deliv Rev, 2013.65(1):36-48.). For example, CN 110638759A discloses an mRNA formulation for in vitro transfection and in vivo delivery. In this invention, the lipid molecules used to bind mRNA are ionizable lipid molecules and cationic lipid molecules. However, these lipid molecules are non-degradable in vivo, posing a potential toxicity. Furthermore, in the practical application of gene vaccines, product stability and storage and transportation have been identified as major bottlenecks. Current gene vaccines have extremely high requirements for storage conditions, needing to be stored at extremely low temperatures, resulting in high application costs and inconvenience.The main factor contributing to formulation instability is the low positive charge density of amine-containing lipids in lipid nanoparticle delivery systems, resulting in weak electrostatic interactions with genes and thus instability and inactivation of lipid nanoparticles.

[0003] Previous studies have shown that high-density cationic polymers or cationic liposomes can effectively compress genetic material. Compared with tertiary amine carriers, cationic carriers containing quaternary ammonium salts can form more stable complexes with genetic material. However, the strong positive charge of quaternary ammonium salt cationic carriers leads to incomplete dissociation of the complex in the cytoplasm, preventing the release of genetic material for transfection and reducing transfection efficiency. Therefore, the high extracellular stability and complete intracellular dissociation of the complex formed by the gene drug and the carrier are contradictory. Publication No. CN 106146834 A discloses an esterase-responsive cationic polymer for preparing DNA complexes for gene transfection. After enzymatic hydrolysis, the high molecular weight polymer backbone of the delivery system is not degraded, exhibiting potential toxicity; furthermore, the tertiary amine structure remains on the polymer backbone, preventing complete dissociation of the nucleic acid drug and reducing the transfection efficiency of the nucleic acid drug. Summary of the Invention

[0004] The purpose of this invention is to solve the problem of XX by providing an esterase-responsive cationic lipid molecule for gene delivery. The liposomes formed by the cationic lipid molecule have good stability in vitro, low toxicity as a carrier in vivo, and can efficiently and completely dissociate from the gene, maximizing protein translation and improving transfection efficiency.

[0005] The technical solution adopted by this invention to solve its technical problem is:

[0006] An esterase-responsive cationic lipid molecule for gene delivery can be hydrolyzed into an electronegative or electronegative compound by intracellular esterases. This compound consists of an X group and n atoms. The compounds formed by the linkage, where n = 1, 2, 3, or 4, have the following structural composition:

[0007]

[0008] R1 is a C1-C4 alkyl or aromatic group, R2 is a C1-C4 alkyl or aromatic group, and R3 is a C5-C30 aliphatic chain or fused ring hydrophobic group.

[0009] X is one of amino group, substituted amino ethyl group, hydroxyl group, ether group, thioether group, and piperazine group.

[0010] Or X is One of them;

[0011] The R4 is one of CH2CH2, CH2CH2CH2, and CH2CH2CH2CH2.

[0012] The fused-ring hydrophobic groups include cholesterol and bile acids.

[0013] The anions are chloride or bromide ions.

[0014] The esterase-responsive cationic lipid molecule is specifically selected from one of the following compounds:

[0015] (1)

[0016] (2)

[0017] (3)

[0018] (4)

[0019] (5)

[0020] (6)

[0021] (7)

[0022] (8)

[0023] (9)

[0024] The esterase-responsive cationic lipid molecules of the present invention can be hydrolyzed by intracellular esterases to remove the positive charge of the quaternary ammonium salt, and then generate a negatively charged carboxylic acid group through autocatalytic ester bond hydrolysis. Taking the above compound (3) as an example, its charge reversal process is shown in the following formula:

[0025]

[0026] Esterase-responsive cationic lipid molecules were prepared by a two-step synthetic method:

[0027] Step 1: First, react the compound containing primary or secondary amine with aminoethyl acrylate in an alcohol solvent at a molar ratio of 1:1-4. The reaction temperature is controlled at 20-80℃ and the reaction time is 12-72h to generate an intermediate product containing a tertiary amine structure.

[0028] Step 2: The intermediate containing the tertiary amine structure is purified by precipitation or column chromatography. Then, the intermediate containing the tertiary amine structure is dissolved in an organic solvent, and one of 4-bromomethylphenol alkyl ester, 4-bromomethylphenol olefinic ester, 4-chloromethylphenol alkyl ester, and 4-chloromethylphenol olefinic ester is added. The mixture is stirred for 12-72 hours, and the reaction temperature is controlled at 20-80℃. After the reaction is completed, the organic solvent is removed, and the esterase-responsive cationic lipid molecule is obtained by precipitation.

[0029] Compounds containing primary or secondary amines include: ethylenediamine, N,N-dimethylethylenediamine, N,N'-dimethylethylenediamine, 1,4-bis(3-propyl)piperazine, N,N-bis(3-propyl)methylamine, N1,N1'-(propane-1,3-diyl)bis(N1-methylethyl-1,2-diamine), and N1-(3-aminopropyl)-N1,N3,N3-trimethylpropyl-1,3-diamine.

[0030] The alcohol solvent is methanol, ethanol, or isopropanol; the organic solvent is one of methanol, N,N-dimethylformamide, and dimethyl sulfoxide.

[0031] Application of esterase-responsive cationic lipid molecules as nucleic acid drug delivery carriers.

[0032] The esterase-responsive cationic lipid molecules of this invention carry a large number of positively charged quaternary ammonium salts, which can form stable nanocomposites with negatively charged nucleic acid drugs through electrostatic self-assembly. After entering the cell, a reaction occurs under the action of the esterase, and the quaternary ammonium salt is converted into a tertiary amine. Subsequently, through autocatalytic hydrolysis of the ester bond, an electrically neutral or negatively charged compound is generated, which repels the nucleic acid drug and rapidly dissociates, completely releasing the nucleic acid drug for efficient transfection.

[0033] This invention also discloses the application of the aforementioned esterase-responsive cationic lipid molecules as gene delivery vectors and their use in gene delivery. The invention prepares a highly efficient, low-toxicity non-viral gene delivery vector, and the nanocomposite formed by this vector and nucleic acid drugs exhibits high transfection efficiency.

[0034] A gene delivery system includes a lipid nanoparticle carrier and a nucleic acid drug loaded on the carrier. The lipid nanoparticle carrier is formed by esterase-responsive cationic lipid molecules, auxiliary lipids, and PEG lipid molecules. The molar ratio of esterase-responsive cationic lipid molecules: auxiliary lipids: PEG lipid molecules is 40-80:10-40:0.5-2.

[0035] The nucleic acid drug is one of DNA, siRNA, shRNA, microRNA, or mRNA.

[0036] The beneficial effects of this invention are as follows: The quaternary ammonium structure has a high cationic charge density, enabling it to tightly bind to DNA or RNA through electrostatic interactions, thus improving the stability of the gene-encapsulating nanoparticles. Furthermore, it allows for complexation at a lower N / P ratio, reducing the amount of lipid molecules used and thus lowering potential toxicity. Under the catalysis of intracellular esterases, the cationic lipid molecules decompose to form non-toxic, electrically neutral or negatively charged molecular fragments, thereby completely releasing the gene for transfection. This delivery system addresses the stability and biosafety issues of gene drug systems while significantly improving gene transfection efficacy, making it suitable for the treatment of related diseases. Attached Figure Description

[0037] Figure 1 The charge reversal potential of cationic lipid molecules in Examples 1-5;

[0038] Figure 2 This is a graph showing the transfection data from the orthogonal experimental ratio screening in Example 4.

[0039] Figure 3 The particle size distribution of the nanoparticles formed from cationic lipid molecules in Examples 1-5 is shown in the diagram.

[0040] Figure 4 Potential diagrams of nanoparticles formed from cationic lipid molecules in Examples 1-5;

[0041] Figure 5 Gene transfection of luciferase protein into nanoparticles formed from cationic lipid molecules, as described in Examples 1-5;

[0042] Figure 6 The data pertain to the cytotoxicity of nanoparticles formed from cationic lipid molecules in Examples 1-5.

[0043] Figure 7 In vivo imaging images of nanoparticles formed by cationic lipid molecules injected intravenously at different times in Example 4;

[0044] Figure 8 This is a comparison of bioluminescence intensity data at different times in vivo after intravenous injection of nanoparticles formed from cationic lipid molecules in Example 4.

[0045] Figure 9 Flow cytometry data comparing the stimulation of DC cell maturation in Examples 3 and 4;

[0046] Figure 10 Example 4 shows the growth inhibition curve of lipid nanoparticles on B16-OVA subcutaneous tumors. Detailed Implementation

[0047] The technical solution of the present invention will be further described in detail below through specific embodiments.

[0048] In this invention, unless otherwise specified, all raw materials and equipment used are commercially available or commonly used in the field. The methods described in the following embodiments are conventional methods in the field, unless otherwise specified.

[0049] definition:

[0050] Representative examples of accessory lipids include, but are not limited to: phosphatidylcholine, distearyl phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, palmitoyl oleoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, dioleoyl phosphatidylcholine, distearyl phosphatidylcholine, dioleoylcholine, trimethyl-2,3-dioleoyloxypropylammonium bromide, trimethyl-2,3-dioleenoyloxypropylammonium chloride, 3β-[N-(N',N'-dimethylaminoethyl)aminoformyl]cholesterol, dioleoyl phosphatidylethanolamine, dimyristoyl phosphatidylcholine, hydrogenated soybean phosphatidylcholine, and dipalmitoyl sphingomyelin.

[0051] Representative examples of PEG lipids include, but are not limited to: dimyristoylglycerol-PEG2000, distearate phosphatidylethanolamine-PEG2000, DMA-PEG2000, and PEG2000-Ceramide-C14.

[0052] Depending on the intended use of the lipid particles, the component ratios can be varied, and the delivery efficiency of the lipid particles can be tested using experiments known in the art.

[0053] Example 1

[0054] Synthesis of compound (1):

[0055] 1 g of N,N,N'-trimethylethylenediamine and 1.4 g of dimethylaminoethyl acrylate were reacted in 10 mL of methanol and stirred at 40 °C for 24 h. After the reaction was complete, the methanol was removed, and the mixture was purified by column chromatography (mobile phase: dichloromethane / methanol = 9 / 1). Then, 1 g of the purified compound was dissolved in 5 mL of N,N-dimethylformamide (DMF), and 0.63 g of 4-bromomethylphenol hexyl ester was added. The mixture was stirred at 20 °C for 24 h. After removing the DMF, the mixture was precipitated with diethyl ether, and the precipitate was collected to give the cationic lipid molecule EMM-PC6 with a yield of 90%. Its structure is as follows:

[0056]

[0057] Example 2

[0058] Synthesis of compound (2):

[0059] 1 g of N,N-dimethylethylenediamine and 3.25 g of dimethylaminoethyl acrylate were stirred in 10 mL of methanol at 60 °C for 48 h. After the reaction was complete, the methanol was removed, and the mixture was purified by column chromatography (mobile phase: dichloromethane / methanol = 95 / 5). Then, 1 g of the purified compound was dissolved in 5 mL of DMF, and 1.73 g of 4-chloromethylphenol dodecyl ester was added. The mixture was stirred at 40 °C for 24 h. After removing the DMF, the mixture was precipitated with diethyl ether, and the precipitate was collected to give the cationic lipid molecule EMTM-PC12 with a yield of 83%. The structure is shown below:

[0060]

[0061] Example 3

[0062] Synthesis of compound (3):

[0063] 1 g of N,N-dimethylethylenediamine and 3.9 g of diethylaminoethyl acrylate were reacted in 10 mL of isopropanol and stirred at 40 °C for 12 h. After the reaction was complete, the isopropanol was removed, and the mixture was purified by column chromatography (mobile phase: dichloromethane / methanol / triethylamine = 90 / 9 / 1). Then, 1 g of the purified compound was dissolved in 5 mL of dimethyl sulfoxide, and 1.74 g of 4-chloromethylphenol dodecyl ester was added. The mixture was stirred at 80 °C for 12 h. After removing the dimethyl sulfoxide, the mixture was precipitated with n-hexane, and the precipitate was collected to give the cationic lipid molecule APB-AC12 with a yield of 71%. Its structure is as follows:

[0064] Example 4

[0065] Synthesis of compound (4):

[0066] 0.36 g of N,N-bis(3-aminopropyl)methylamine and 2.27 g of dibutylaminoethyl acrylate were reacted in 10 mL of isopropanol and stirred at 50 °C for 72 h. After the reaction was complete, methanol was removed, and the mixture was purified by column chromatography (mobile phase: dichloromethane / methanol = 20 / 1). Then, 0.2 g of the purified compound was dissolved in 5 mL of DMF. Next, 0.45 g of 4-bromomethylphenol ester was added, and the mixture was stirred at 80 °C for 12 h. DMF was removed, and the mixture was precipitated with n-hexane. The precipitate was collected to give the cationic lipid molecule AMB-P at °C 18, with a yield of 86%. Its structure is as follows:

[0067]

[0068] Example 5

[0069] Synthesis of compound (5):

[0070] 0.5 g of N-methylethylenediamine and 3.47 g of diethylaminoethyl acrylate were stirred in 10 mL of ethanol at 20 °C for 24 h. After the reaction was complete, methanol was removed, and the mixture was purified by column chromatography (mobile phase: dichloromethane / methanol = 20 / 1). Then, 0.2 g of the purified compound was dissolved in 5 mL of DMSO, and 0.46 g of 4-bromomethylphenol octadecyl ester was added. The mixture was stirred at 70 °C for 48 h. DMSO was removed, and the mixture was precipitated with n-hexane. The precipitate was collected to give the cationic lipid molecule DMB-AC18 in 64% yield. Its structure is as follows:

[0071] Application Example 1

[0072] Porcine liver esterase was dissolved in a 100mM Tris-HCl buffer solution containing 100mM NaCl to prepare a stock solution with an enzyme concentration of 120 units / mL. The cationic lipid compounds from Examples 1-5 were dissolved in DMSO to prepare a solution with a concentration of 30 mg / mL. 0.2 mL of this solution was added to 1.0 mL of the esterase solution. The solution was placed in a 37°C incubator. 100 μL samples were taken at different time points and added to 1 mL of acetonitrile to terminate the enzymatic reaction. The solution was centrifuged at 6000 rpm for 5 min, and the supernatant was collected for potential measurement.

[0073] according to Figure 1 As shown, the cationic lipid molecules in Examples 1-5, under the action of esterases, all exhibited a decrease in potential to neutral or negative over time. This indicates that they can undergo charge reversal under physiological conditions, which facilitates gene dissociation and reduces carrier toxicity.

[0074] Application Example 2

[0075] Nanoparticles were prepared according to Examples 1-5: Cationic lipids, auxiliary lipids, and PEG lipids were dissolved in ethanol solution according to the prescribed amounts to obtain an organic phase with a total concentration of 1 mg / mL. mRNA was dissolved in citrate-sodium citrate buffer (pH = 3, concentration 10 mM) to obtain an aqueous phase with a concentration of 1 mg / mL. Equal volumes of both were mixed, vortexed, and dialyzed in 20 mM MES buffer (pH = 5). After 1 h, a lipid nanoparticle solution was obtained.

[0076] Optimal formulation ratio determination: Utilizing enhanced green fluorescent protein mRNA (EGFP) as a reporter gene to improve in vitro cell transfection efficiency, the optimal N / P ratio was determined by mixing cationic lipid molecules and mRNA molecules at different N / P ratios in HeLa cell transfection experiments, as described in Example 4. Based on this N / P ratio, a three-factor, four-level orthogonal experiment was designed to screen for the best ratio in HeLa cell transfection experiments. The three factors included: cationic lipid molecules, helper lipids (e.g., DOPE), and PEG lipids (e.g., DMG-PEG2000), all expressed in moles.

[0077] cationic lipid molecules DOPE DMG-PEG 1 50 10 0.5 2 50 20 1 3 50 30 1.5 4 50 40 2 5 60 10 1 6 60 20 0.5 7 60 30 2 8 60 40 1.5 9 70 10 1.5 10 70 20 2 11 70 30 0.5 12 70 40 1 13 80 10 2 14 80 20 1.5 15 80 30 1 16 80 40 0.5

[0078] Results analysis is as follows: Figure 2 As shown, based on the mean main effect analysis, the optimal ratio for this orthogonal experiment is cationic lipid molecules: DOPE: DMG-PEG2000 molar ratio = 50:20:0.5. This ratio is also used for the proportion of nanoparticle components in other application examples.

[0079] Application Example 3

[0080] In Examples 1-5, cationic lipid molecules were used to prepare lipid nanoparticles according to a molar ratio of cationic lipid molecules:DOPE:DMG-PEG2000 = 50:20:0.5. Particle size potential was measured: the size and zeta potential of the prepared lipid nanoparticle solution were measured using a dynamic light scattering instrument at 25°C. The results were calculated using DTS software. Each group was repeated three times, and the average value was taken. Figure 3 and Figure 4 The nanoparticles have a particle size of 90-250 nm, a PDI of less than 0.25, and a potential between -5 and 15 mV.

[0081] Application Example 4

[0082] Cell transfection assay of luciferase mRNA from lipid nanoparticles: HeLa cells were cultured in 96-well plates at a density of 20,000 cells / well, with 0.4 mL of culture medium per well. Cells were incubated at 37°C for 24 h in a 5% CO2, 95% humidity incubator. Afterward, the culture medium in each well was replaced with 0.4 mL of the prepared lipid nanoparticle solution (containing 0.1 μg luciferase mRNA), and cultured for another 24 h. Dlin-MC3-DMA ionizable lipid molecules and commercially available liposomes were used as control groups. The culture medium was discarded, and 100 μL of cell lysis buffer was added to each well. After complete cell lysis, luciferase substrate was added, and the chemiluminescence intensity was measured using a chemiluminescence detector. Protein concentration was determined using the Bradford protein assay kit. Chemiluminescence intensity was normalized to protein concentration and is expressed as luminescence intensity per milligram of protein (RLU / mg protein). Each data set represents the average of three wells in the same sample group.

[0083] Figure 5 The transfection of the cationic lipid nanoparticles from Examples 1-5 into HeLa cells is shown. The nanoparticles from Examples 2, 4, and 5 showed higher luciferase protein expression in HeLa cells compared to the commercially available Dlin-MC3-DMA liposomes. Furthermore, compared to polymer A1 in Publication No. CN 106146834 A, under an N / P ratio of 20, their mRNA-protein translation efficiency was weaker than that of the cationic lipid nanoparticles from Examples 1-5.

[0084] Application Example 5

[0085] Cytotoxicity assays of cationic lipid molecules and corresponding lipid nanoparticles: 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide (MTT) was used to detect the in vitro cytotoxicity of cationic lipid molecules and corresponding lipid nanoparticles in HeLa cells. Cells were cultured in 96-well plates at a density of 5000 cells / well, with 180 μL of culture medium added to each well. Cells were incubated for 24 h at 37°C under 5% CO2 and 95% humidity. After 24 h, 20 μL of different concentrations (concentration gradients were determined according to the molar amount of cationic lipid molecules required for a specific N / P ratio) of lipid molecule culture medium or lipid nanoparticle culture medium was added to each well. A blank control group received 20 μL of culture medium. Cells were cultured for another 48 h. The complex formed by Lipofectamine 2000 and mRNA served as a control group. After culturing for 48 h, 20 μL of MTT solution (5 mg / mL) was added and incubated at 37 °C for 4 h. The cells were then centrifuged at 2500 rpm for 5 min, discarding all MTT culture medium from each well. 100 μL of DMSO was added, and the mixture was shaken to dissolve all crystals in each well. The absorbance of the samples at 562 and 620 nm was measured using a microplate reader. Cell viability (percentage) was expressed as the absorbance value of the experimental group divided by the absorbance value of the control group. All data for each group are the average of three wells of the same sample.

[0086] Figure 6 The data provided are for the cytotoxicity of the nanoparticles composed in Examples 1-5. Within the normal concentration range of nanoparticles, the cytotoxicity is low, and all are lower than the cytotoxicity of the complex formed by Lipofectamine 2000.

[0087] Application Example 6

[0088] In vivo transfection organ distribution and delivery efficiency: Referring to the preparation process of Application Example 2, the cationic lipids in Example 4 were selected to prepare lipid nanoparticles in the optimal ratio. 5 μg of reporter gene luciferase mRNA was delivered to mice via tail vein injection. The luciferase substrate was injected into the mice at 1 h, 3 h, 6 h, 12 h, and 24 h. The mice were anesthetized and dissected 10 min later. The expression site and intensity changes of luciferase in mice were observed using a fluorescence in vivo imaging system.

[0089] pass Figure 7 Observation of the bioluminescence intensity of luciferase at different time points indicated that the lipid nanoparticles were highly expressed in the spleen and had a spleen-targeted enrichment effect. Figure 8 Quantitative bioluminescence analysis showed that the luminescence intensity reached its peak 6 hours after intravenous injection of the lipid nanoparticles. This indicates that this series of lipid nanoparticles can be used to treat spleen-related diseases and immune disorders.

[0090] Application Example 7

[0091] Mouse bone marrow mesenchymal stem cells were extracted to induce BMDC cells. Cells were collected on day 6 and cultured at 5 × 10⁶ cells per well. 5 Cells were added to 24-well plates at a specific ratio. After 24 hours, the cells were incubated for 24 hours in complete medium with different LNP formulations (containing 0.5 μg OVA mRNA) from Examples 3 and 4. LPS served as a positive control. After co-incubation, BMDCs were collected, washed with FACS buffer, incubated with an Fc receptor blocker at room temperature, and then stained on ice with fluorophore-labeled anti-CD11c, CD80, and CD86 antibodies. Flow cytometry was used to quantify the labeled cells.

[0092] pass Figure 9 Data show that the lipid nanoparticles formed by the cationic lipid molecules in Examples 3 and 4 in the optimal ratio have a higher ability to stimulate BMDC maturation than the positive control group, indicating that the lipid nanoparticles can effectively transfect BMDC cells and exert immune function.

[0093] Application Example 13

[0094] Lipid nanoparticles, formed from cationic lipid molecules in the optimal ratio as described in Example 4, were loaded with 5 μg of OVA-mRNA. C57bl / 6 mice were subcutaneously injected with 300,000 B16-OVA cells, and the lipid nanoparticles were injected on days 4 and 9, respectively. An equal volume of PBS was injected as a negative control. The MC3 liposome group and the OVA protein group, with their publicly disclosed formulations, were used as control groups. Figure 10 The data show that the lipid nanoparticle group effectively inhibited tumor growth, with tumors remaining small on day 22 and no mice dying; while the OVA protein group and MC3 liposome group both showed tumor volumes greater than 2000 cm³ on days 16 and 20. 3 The situation indicates that the tumor-suppressing effect is poor.

[0095] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the present invention in any way. Other variations and modifications are possible without departing from the technical solutions described in the claims.

Claims

1. An esterase-responsive cationic lipid molecule for gene delivery, characterized in that, which can be hydrolyzed into electrically neutral or negatively charged compounds by intracellular esterase, and the esterase responds to cationic lipid molecules, and the esterase responds to cationic lipid molecules are specifically selected from one of the following compounds: ； R1 is a C1-C4 alkyl, R2 is a C1-C4 alkyl, and R3 is a C5-C30 aliphatic chain; R4 is one of CH2CH2, CH2CH2CH2, and CH2CH2CH2CH2.

2. The esterase-responsive cationic lipid molecule for gene delivery according to claim 1, characterized by, The anion of the esterase responding to the cationic lipid molecule is chloride or bromide.

3. The esterase-responsive cationic lipid molecule for gene delivery according to claim 1, characterized by, The esterase responding to the cationic lipid molecule is prepared by a two-step synthesis method: First step: first, the compound containing a primary amine or a secondary amine is stirred with aminoethyl acrylate in an alcohol solvent at a molar ratio of 1:1-4, the stirring reaction temperature is controlled at 20-80°C, and the stirring reaction time is 12-72h to generate an intermediate product containing a tertiary amine structure; Second step: the intermediate product containing a tertiary amine structure is purified by precipitation or column chromatography, then the intermediate product containing a tertiary amine structure is dissolved in an organic solvent, and one of 4-bromomethylphenol alkyl ester, 4-bromomethylphenol alkenyl ester, 4-chloromethylphenol alkyl ester, and 4-chloromethylphenol alkenyl ester is added, and the stirring reaction is carried out for 12-72h, the reaction temperature is controlled at 20-80°C, and after the reaction is completed, the organic solvent is removed, and the esterase responding to the cationic lipid molecule is obtained by purification by precipitation.

4. The esterase-responsive cationic lipid molecule for gene delivery according to claim 3, characterized by, The compound containing a primary amine or a secondary amine includes ethylenediamine, N,N-dimethylethylenediamine, and N,N'-dimethylethylenediamine.

5. The esterase-responsive cationic lipid molecule for gene delivery according to claim 3, characterized by, The alcohol solvent is methanol, ethanol, or isopropanol; and the organic solvent is one of methanol, N,N-dimethylformamide, and dimethyl sulfoxide.

6. The esterase responding to the cationic lipid molecule of claim 1 as a nucleic acid drug delivery carrier.

7. A gene delivery system, characterized by, The nucleic acid drug delivery carrier comprises a lipid nanoparticle carrier and a nucleic acid drug loaded on the carrier, and the lipid nanoparticle carrier is formed by the esterase responding to the cationic lipid molecule, the auxiliary lipid, and the PEG lipid molecule; the molar ratio of the esterase responding to the cationic lipid molecule, the auxiliary lipid, and the PEG lipid molecule is 40-80:10-40:0.5-2.

8. The gene delivery system of claim 7, wherein, The nucleic acid drug is one of DNA, siRNA, shRNA, micro RNA, and mRNA.

9. An esterase-responsive cationic lipid molecule for gene delivery, characterized in that, The esterase responding to the cationic lipid molecule is specifically a compound with the following structure: 。

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