Nucleic acid delivery system as well as preparation method and application thereof

Abstract

The invention discloses a nucleic acid delivery system and a preparation method and application thereof, and belongs to the technical field of biological materials. According to the invention, a benzenesulfonamide functional group is introduced into a GSs molecular skeleton, such that a novel benzenesulfonamide functionalized gemini surfactant (NGS) is constructed. Wherein benzenesulfonamide is a specific endoplasmic reticulum targeting group, and can realize endoplasmic reticulum targeting enrichment through specific binding with a sulfonylurea receptor on an endoplasmic reticulum membrane, so that the NGS gene delivery vector is distributed towards the endoplasmic reticulum. After the NGS and the pDNA form a compound, the compound enters cells through cavette protein mediated endocytosis, then targets the endoplasmic reticulum and releases the pDNA, the pDNA is delivered into the cell nucleus by utilizing the close relation between the endoplasmic reticulum and the nuclear membrane, the gene transfection efficiency is finally improved, meanwhile, the biocompatibility is further improved by introducing the gamma-polyglutamic acid (gamma-PGA) with negative charges, and the application prospect is wide.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials technology, specifically relating to a nucleic acid delivery system, its preparation method, and its application. Background Technology

[0002] In recent years, gene therapy has shown broad application prospects in cancer, infectious diseases, genetic diseases, and rare diseases. Its core lies in delivering exogenous nucleic acids such as plasmid DNA (pDNA), messenger RNA (mRNA), and small interfering RNA (siRNA) to regulate or repair abnormal gene expression, thereby achieving precise treatment of diseases. However, nucleic acids themselves are characterized by large molecular weight, negative charge, and poor stability. During in vivo delivery, multiple extracellular and intracellular barriers must be overcome, including degradation by nucleases in the blood, clearance by the reticuloendothelial system, electrostatic repulsion of the cell membrane, retention by intracellular lysosomes, and restricted nuclear entry. Therefore, constructing an efficient and safe nucleic acid delivery system is crucial for the success of gene therapy.

[0003] Non-viral vector systems, as an advanced nucleic acid delivery method, have attracted widespread research and attention compared to viral vectors due to their advantages such as low immunogenicity, strong structural tunability, and lower preparation cost. Among them, cationic gemini surfactants (GSs), formed by two monomeric surfactants linked by a spacer group, possess high charge density and excellent nucleic acid compression ability. They can form small-particle, positively charged nanocomposites with nucleic acids through electrostatic interactions, making them one of the important research directions in current non-viral delivery systems. However, existing GS gene delivery systems still face challenges such as low transfection efficiency due to lysosomal retention during intracellular transport or nuclear membrane barrier limitations, restricting their clinical translation. Furthermore, biomimetic modifications based on cell membranes may present problems in terms of sourcing, large-scale production, and long-term storage, severely limiting the clinical application of GS delivery vectors. Therefore, there is an urgent need to rationally design next-generation GS-based nucleic acid delivery vectors to achieve a balance between high transfection efficiency and low cytotoxicity. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a nucleic acid delivery system, its preparation method and application, so as to solve the technical problems of low transfection efficiency and poor delivery effect of the existing GSs gene delivery system.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is: to provide a method for preparing a nucleic acid delivery system, comprising the following steps: S1. 2-(4-aminosulfonylphenyl)acetic acid and N,N′-carbonyldiimidazole are mixed and dissolved, and then stirred for 20-40 min. 2-(4-aminosulfonylphenyl)acetic acid provides the carboxyl group (-COOH) as the active site, and N,N′-carbonyldiimidazole acts as a condensing agent to react with the carboxyl group of 2-(4-aminosulfonylphenyl)acetic acid to generate an acylimidazole intermediate. Then, a nucleophilic amine reactant, 3,3′-iminobis(N,N-dimethylpropylamine), is added and mixed evenly. The mixture is then refluxed at 60-80℃ with the activated acylimidazole intermediate for 1-3 h to undergo an amidation reaction to form an amide bond, yielding an intermediate containing a benzenesulfonamide group. The molar ratio of 2-(4-aminosulfonylphenyl)acetic acid, N,N′-carbonyldiimidazole, and 3,3′-iminobis(N,N-dimethylpropylamine) is 1-2:1-2:1-2. S2. The intermediate product and bromooctadecane are mixed and dissolved in a molar ratio of 0.5-1.5:2-3, and then refluxed at 90-110℃ for 70-74h. Bromooctadecane, as a long-chain alkylating agent, undergoes a typical nucleophilic substitution reaction (Menshutkin reaction) with the tertiary amine group in the intermediate product to generate a quaternary ammonium salt structured gemini surfactant (NGS). The bisquaternary ammonium salt structure significantly enhances the electrostatic interaction and compressibility between NGS and negatively charged nucleic acid molecules. At the same time, the two long-chain hydrophobic alkyl tail chains introduced are beneficial to maintaining the stability of the nanostructure. S3. Mix the Gemini surfactant (NGS) and the substance to be delivered at a mass ratio of 6-10:0.5-1.5, repeatedly blow and vortex for 5-10 seconds, and then incubate at room temperature for 0.5-1.5 hours to obtain NGS nanoparticles; the substance to be delivered is plasmid DNA (pDNA), messenger RNA (mRNA), small interfering RNA (siRNA), or antisense oligonucleotide.

[0006] Based on the above technical solution, the present invention can be further improved as follows: Furthermore, the solvents used for dissolving in S1 and S2 are tetrahydrofuran or acetonitrile.

[0007] Furthermore, the molar ratio of 2-(4-aminosulfonylurea)acetic acid, N,N′-carbonyldiimidazole and 3,3′-iminobis(N,N-dimethylpropylamine) is 1:1:1.

[0008] Furthermore, it also includes: mixing a negatively charged γ-polyglutamic acid solution and NGS nanoparticles at a ratio of 1.9-9.7 μL: 15-20 μg, repeatedly blowing and agitating 3-5 times, and then incubating at room temperature for 30-60 min to obtain NGS-PGA nanoparticles by electrostatic bonding.

[0009] Furthermore, in NGS nanoparticles, N:P = 1-6:1; in NGS-PGA nanoparticles, N:P:C = 5:1:0.25-1.25, where N represents the number of moles of protonated amino groups in the NGS molecule, P represents the number of moles of phosphate groups in the nucleic acid, and C represents the number of moles of carboxyl groups in γ-polyglutamic acid.

[0010] Furthermore, the concentration of the γ-polyglutamic acid solution was 90-110 μg / mL.

[0011] The present invention also discloses a nucleic acid delivery system (NGS nanoparticles or NGS-PGA nanoparticles) prepared by the above preparation method.

[0012] The present invention also discloses the application of the above-mentioned nucleic acid delivery system in the preparation of nucleic acid delivery drugs.

[0013] The beneficial effects of this invention are as follows: 1. This invention constructs a novel benzenesulfonamide-functionalized gemini surfactant (NGS) by introducing a benzenesulfonamide functional group into the GS molecular backbone. The benzenesulfonamide group is a specific endoplasmic reticulum (ER) targeting group, which can achieve ER-targeted enrichment through specific binding to sulfonylurea receptors on the ER membrane, thereby distributing the NGS gene delivery vector to the ER. After NGS forms a complex with pDNA, it enters the cell via caveolin-mediated endocytosis, then targets the ER and releases pDNA. Utilizing the close connection between the ER and the nuclear membrane, the pDNA is delivered to the nucleus, ultimately improving gene transfection efficiency. Simultaneously, the introduction of negatively charged γ-polyglutamate (γ-PGA) further improves biocompatibility.

[0014] 2. This invention employs an NGS-mediated nucleic acid loading method, which offers high encapsulation efficiency, simple and rapid operation, and effectively prevents pDNA degradation by nucleases. Compared to unmodified benzenesulfonamide-based GS, the NGS nanoparticles and NGS-PGA nanoparticles prepared in this invention exhibit higher cellular uptake, transfection efficiency, and biocompatibility as nucleic acid delivery systems.

[0015] 3. This invention uses γ-PGA-modified NGS-PGA nanoparticles as nucleic acid delivery carriers, without involving problems such as sourcing, large-scale production, and long-term storage caused by cell membrane modification. γ-PGA is directly incubated with NGS nanoparticles to form a low-cost nucleic acid delivery material that can be mass-produced. Attached Figure Description

[0016] Figure 1 This is a synthetic route diagram of the gemini surfactant (NGS) of the present invention; Figure 2 The synthetic route of Gemini surfactant (GS) in Comparative Example 1 is shown. Figure 3Results of gel retardation experiments for NGS nanoparticles with different N / P ratios; Figure 4 The gel retardation experiment results for NGS-PGA nanoparticles (1w) with different N / P / C ratios; Figure 5 The gel retardation experiment results are for NGS-PGA nanoparticles (30w) with different N / P / C ratios; Figure 6 This is a particle size distribution diagram of NGS nanoparticles; Figure 7 The particle size distribution of NGS-PGA nanoparticles (1w) is shown. Figure 8 The particle size distribution of NGS-PGA nanoparticles (30w) is shown. Figure 9 Transmission electron microscopy images of NGS nanoparticles, NGS-PGA nanoparticles (1w), and NGS-PGA nanoparticles (30w); Figure 10 Transfection diagram of AML-12 cells with GS-pDNA and NGS-PGA nanoparticles (1w); Figure 11 Quantitative diagram of GS-pDNA and NGS-PGA nanoparticles (1w); Figure 12 Image showing AML-12 cell transfection with GS-pDNA and NGS-PGA nanoparticles (30w); Figure 13 Quantitative diagrams of GS-pDNA and NGS-PGA nanoparticles (30w); Figure 14 In vivo transfection diagrams of GS-pDNA, NGS nanoparticles, NGS-PGA nanoparticles (1w), and NGS-PGA nanoparticles (30w) at treatment times of 24, 48, and 72 h; Figure 15 Quantitative diagrams of GS-pDNA, NGS nanoparticles, NGS-PGA nanoparticles (1w), and NGS-PGA nanoparticles (30w) at treatment times of 24, 48, and 72 h; Figure 16 Hemolysis diagrams of GS-pDNA, NGS nanoparticles, NGS-PGA nanoparticles (1w), and NGS-PGA nanoparticles (30w) at treatment times of 1, 5, and 24 h. Figure 17 Quantitative diagrams of GS-pDNA, NGS nanoparticles, NGS-PGA nanoparticles (1w), and NGS-PGA nanoparticles (30w) at treatment times of 1, 5, and 24 h. Figure 18Cell viability graph of AML-12 cells after 24 h of treatment with GS-pDNA and NGS-PGA nanoparticles with different N / P / C ratios (1w); Figure 19 Cell viability graph of AML-12 cells after 24 h of treatment with GS-pDNA and NGS-PGA nanoparticles (30w) with different N / P / C ratios; Figure 20 Confocal images of NGS nanoparticles, NGS-PGA nanoparticles (1w), and NGS-PGA nanoparticles (30w); Figure 21 Quantitative diagrams of NGS nanoparticles, NGS-PGA nanoparticles (1w), and NGS-PGA nanoparticles (30w); Figure 22 AML-12 cell uptake of GS-pDNA, NGS nanoparticles, NGS-PGA nanoparticles (1w), and NGS-PGA nanoparticles (30w); Figure 23 Quantitative diagrams of GS-pDNA, NGS nanoparticles, NGS-PGA nanoparticles (1w), and NGS-PGA nanoparticles (30w); Figure 24 Particle size variation of NGS nanoparticles, NGS-PGA nanoparticles (1w), and NGS-PGA nanoparticles (30w) in 10% fetal bovine serum; Figure 25 The graph shows the variation of the dispersion index (PDI) of NGS nanoparticles, NGS-PGA nanoparticles (1w), and NGS-PGA nanoparticles (30w) in 10% fetal bovine serum. Detailed Implementation

[0017] The specific embodiments of the present invention are described below to facilitate understanding of the invention by those skilled in the art. Unless otherwise specified, specific conditions are applied according to conventional conditions or the manufacturer's recommendations. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various modifications are obvious as long as they fall within the spirit and scope of the invention as defined and determined by the appended claims. All inventions utilizing the concept of this invention are protected.

[0018] The plasmid DNA (pDNA) used in the following embodiments is pEGFP-C1 plasmid (4731bp), a commonly used eukaryotic expression reporter gene, containing the following key elements: CMV promoter and enhancer (driving EGFP expression), EGFP reporter gene, multiple cloning site (including XhoI and BamHI sites), AmpR and NeoR / KanR resistance genes, SV40 and HSV TK poly(A) transcription termination signals, common origin of replication (ori) and f1 ori, purchased from Wuhan Miaoling Biotechnology Co., Ltd., product code P0134.

[0019] Example 1 A method for preparing a nucleic acid delivery system includes the following steps: The synthetic route diagram for S1 and Gemini surfactants (NGS) is as follows: Figure 1 As shown, 2-(4-aminosulfonylphenyl)acetic acid (substance A) and N,N′-carbonyldiimidazole were mixed and dissolved in tetrahydrofuran, and then stirred for 30 min. Next, 3,3′-iminobis(N,N-dimethylpropylamine) was added, mixed thoroughly, and refluxed at 70 °C for 2 h to obtain the intermediate product (substance C). The molar ratio of 2-(4-aminosulfonylphenyl)acetic acid, N,N′-carbonyldiimidazole, and 3,3′-iminobis(N,N-dimethylpropylamine) was 1:1:1. S2. The intermediate product and bromooctadecane were mixed and dissolved in acetonitrile at a molar ratio of 1:2.5, and then refluxed at 100°C for 72 h. After the reaction was completed, the mixture was recrystallized and filtered through acetonitrile / ethanol (volume ratio of 1:1) to obtain a white powdered Gemini surfactant (NGS, substance D). S3. Disperse and dissolve the Gemini surfactant (NGS) and plasmid DNA (pDNA) separately. Then, add 2 μL of pDNA solution with a concentration of 1 μg / μL to 15.9 μL of NGS solution with a concentration of 1 μg / μL. Repeat the pipetting and vortexing process 6 times and 8 seconds. Then, incubate at room temperature for 1 hour to obtain NGS nanoparticles (N / P ratio of 5:1). S4. Mix 7.7 μL of γ-polyglutamic acid (molecular weight 10 kDa) solution with 100 μg / mL and 17.9 μg of NGS nanoparticles, repeat the mixing 4 times, and then incubate at room temperature for 45 min to obtain a nucleic acid delivery system (NGS-PGA nanoparticles (1w)) with an N / P / C ratio of 5:1:1.

[0020] Example 2 The difference between this embodiment and Example 1 is that the γ-polyglutamic acid with a molecular weight of 10kDa in S4 is replaced with γ-polyglutamic acid with a molecular weight of 300kDa. The other implementation conditions are the same as in Example 1, and a nucleic acid delivery system (NGS-PGA nanoparticles (30w)) with an N / P / C ratio of 5:1:1 is obtained.

[0021] Example 3 A method for preparing a nucleic acid delivery system includes the following steps: The synthetic route diagram for S1 and Gemini surfactants (NGS) is as follows: Figure 1 As shown, 2-(4-aminosulfonylphenyl)acetic acid and N,N′-carbonyldiimidazole were mixed and dissolved in tetrahydrofuran, and then stirred for 20 min. Next, 3,3′-iminobis(N,N-dimethylpropylamine) was added, mixed thoroughly, and refluxed at 80 °C for 1 h to obtain the intermediate product. The molar ratio of 2-(4-aminosulfonylphenyl)acetic acid, N,N′-carbonyldiimidazole, and 3,3′-iminobis(N,N-dimethylpropylamine) was 1:2:1. S2. The intermediate product and bromooctadecane were mixed and dissolved in acetonitrile at a molar ratio of 0.5:2, and then refluxed at 90°C for 74 h. After the reaction was completed, the mixture was recrystallized and filtered through acetonitrile / ethanol (volume ratio of 1:1) to obtain a white powder of Gemini surfactant (NGS). S3. Disperse and dissolve the Gemini surfactant (NGS) and plasmid DNA (pDNA) separately. Then add 2 μL of pDNA solution with a concentration of 1 μg / μL to 15.9 μL of NGS solution with a concentration of 1 μg / μL. Repeat the pipetting and vortexing 5 times and 10 s. Then incubate at room temperature for 1.5 h to obtain NGS nanoparticles.

[0022] Example 4 A method for preparing a nucleic acid delivery system includes the following steps: The synthetic route diagram for S1 and Gemini surfactants (NGS) is as follows: Figure 1 As shown, 2-(4-aminosulfonylphenyl)acetic acid and N,N′-carbonyldiimidazole were mixed and dissolved in tetrahydrofuran, and then stirred for 40 min. Next, 3,3′-iminobis(N,N-dimethylpropylamine) was added, mixed thoroughly, and refluxed at 60 °C for 3 h to obtain the intermediate product. The molar ratio of 2-(4-aminosulfonylphenyl)acetic acid, N,N′-carbonyldiimidazole, and 3,3′-iminobis(N,N-dimethylpropylamine) was 2:1:2. S2. The intermediate product and bromooctadecane were mixed and dissolved in acetonitrile at a molar ratio of 1.5:2, and then refluxed at 110°C for 70 h. After the reaction was completed, the mixture was recrystallized and filtered through acetonitrile / ethanol (volume ratio of 1:1) to obtain a white powdered Gemini surfactant (NGS). S3. Disperse and dissolve the Gemini surfactant (NGS) and plasmid DNA (pDNA) separately. Then add 2 μL of pDNA solution with a concentration of 1 μg / μL to 15.9 μL of NGS solution with a concentration of 1 μg / μL. Repeat the pipetting and vortexing process 8 times and 5 seconds. Then incubate at room temperature for 0.5 h to obtain NGS nanoparticles. S4. Mix 7.7 μL of γ-polyglutamic acid (molecular weight 300 kDa) solution with 110 μg / mL and 17.9 μg of NGS nanoparticles, repeat the mixing three times, and then incubate at room temperature for 30 min to obtain the nucleic acid delivery system (NGS-PGA nanoparticles (30w)).

[0023] Comparative Example 1 A method for preparing a GS-pDNA nucleic acid delivery system, the synthetic route of the gemini surfactant (GS) is shown below. Figure 2 As shown, the process includes the following steps: N,N,N',N'-tetramethyl-1,7-heptanediamine (0.93 g, 5.0 mmol) was dissolved in 30 mL of acetonitrile, and bromooctadecane (3.51 g, 10.5 mmol) was added under stirring. The reaction system was heated to 100 °C, then refluxed and stirred continuously for 12 h. After the reaction was completed, the mixture was cooled to room temperature, and the resulting solid was collected by vacuum filtration and recrystallized from acetonitrile to obtain the white solid product GS. The subsequent preparation steps are the same as step S3 in Example 1, to obtain the GS-pDNA nucleic acid delivery system.

[0024] Experiment Example 1: Nucleic Acid Packaging 1. Taking the preparation method of Example 1 as an example, by adjusting the amount of gemini surfactant (NGS) and plasmid DNA (pDNA) in step S3, NGS nanoparticles with different N / P ratios (N / P ratios of 1:1, 2:1, 3:1, 4:1, 5:1 and 6:1) were obtained; then, NGS nanoparticles with an N / P ratio of 5:1 were used to continue preparation, and the amount of γ-polyglutamic acid and NGS nanoparticles in step S4 was adjusted to obtain NGS-PGA nanoparticles (1w) with different N / P / C ratios (N / P / C ratios of 5:1:0, 5:1:0.25, 5:1:0.5, 5:1:0.75, 5:1:1 and 5:1:1.25).

[0025] Taking the preparation method of Example 2 as an example, by adjusting the amount of γ-polyglutamic acid and NGS nanoparticles in step S4, NGS-PGA nanoparticles (30w) with different N / P / C ratios (N / P / C ratios of 5:1:0, 5:1:0.25, 5:1:0.5, 5:1:0.75, 5:1:1 and 5:1:1.25) were obtained.

[0026] 2. Weigh 400 mg of agarose powder, add 40 mL of TAE buffer to the agarose powder, heat to dissolve, and cool to obtain a 1% (w / w) agarose solution. Then, add NGS nanoparticles with different N / P ratios and NGS-PGA nanoparticles with different N / P / C ratios to the sample wells. Electrophoresis is performed in a bedroom electrophoresis apparatus containing TAE buffer under constant voltage of 130 V for 50 min.

[0027] The results are as follows Figures 3-5 As shown, when the N / P ratio of NGS to pDNA is 5:1, no electrophoretic bands appear at all, and the pDNA complexed by NGS is retained in the gel wells, meaning that NGS completely encapsulates the pDNA. Figure 3 Furthermore, under the condition that the N / P ratio of NGS to pDNA is 5:1, as the C / P ratio of γ-PGA to pDNA varied from 0.25 to 1.25, none of the NGS-PGA nanoparticles (10kDa / 300kDa) formed with different molecular weights of γ-PGA showed any electrophoretic bands for pDNA. This indicates that NGS can still completely condense pDNA, therefore the condensation of pDNA by NGS is not affected by the addition of γ-PGA. Figure 4 and Figure 5 ).

[0028] Experimental Example 2: Material Characterization 1. Particle size and zeta potential NGS nanoparticles (N / P ratio 5:1), NGS-PGA nanoparticles (10kDa) (N / P / C ratio 5:1:1), and NGS-PGA nanoparticles (300kDa) (N / P / C ratio 5:1:1) were diluted 50 times with ultrapure water. The particle size and potential of each sample were measured at 25℃ using a Malvern particle size potential analyzer. Each group was measured 3 times, with 11 rounds each time.

[0029] The results are as follows Figures 6-8 As shown, the average particle size of all the complexes is less than 200 nm, and the potential of the NGS nanoparticles decreased to 19.5 mV and 18.9 mV, respectively, after the addition of γ-PGA of different molecular weights (10 kDa and 300 kDa).

[0030] 2. Nanoparticle morphology The morphology and size of NGS-based ternary and binary complexes were observed using transmission electron microscopy (TEM). Figure 9 As shown, all nanocomposites (NGS nanoparticles, NGS-PGA nanoparticles (10kDa) (N / P / C ratio of 5:1:1), NGS-PGA nanoparticles (300kDa) (N / P / C ratio of 5:1:1)) have relatively uniform particle size distribution, with particle size ranging from 100 to 200 nm, and exhibit a regular and full spherical structure.

[0031] Experiment Example 3: Cell Transfection Efficiency AML-12 cells were loaded at 5 × 10 5 Cells were seeded in 6-well plates using DMEM / F12 medium, 10% fetal bovine serum, 1% ITS (insulin-transferrin-selenium supplement), and 40 ng / mL dexamethasone. The cells were then cultured overnight at 37°C with 5% CO2. When cell confluence reached approximately 80%, GS-pDNA (Comparative Example 1) and different proportions of NGS-PGA nanoparticles (10 kDa) prepared in Experiment 1 were added to the cells under fetal bovine serum-free conditions and incubated for 4 h. Lipo2000-pDNA was used as a positive control. After incubation, the culture medium containing the transfection complex was aspirated, the cells were washed twice with PBS, and 2 mL / well of fresh AML-12 culture medium containing 10% FBS was added. The cells were cultured for another 24 h, and finally, fluorescence expression in the cells was observed using an inverted fluorescence microscope.

[0032] like Figures 10-13 As shown, the transfection efficiency of the NGS-PGA nanoparticle group is significantly stronger than that of GS-pDNA, with the transfection efficiency of NGS nanoparticles = 5:1 being 33 times that of GS-pDNA.

[0033] The N / P ratio of the NGS nanoparticles used in subsequent experiments was 5:1, and the N / P / C ratio of both the NGS-PGA nanoparticles (10kDa) and NGS-PGA nanoparticles (300kDa) was 5:1:1.

[0034] Experiment Example 4: Animal Experiment 1. In vivo transfection efficiency BALB / c mice were used as experimental animals to establish a tail vein drug administration model. Transfection complexes containing EGFP fluorescent protein-encoding plasmids were prepared as GS-pDNA, NGS nanoparticles, NGS-PGA nanoparticles (10 kDa), and NGS-PGA nanoparticles (300 kDa), with each complex containing 30 μg of EGFP plasmid. Mice were sacrificed at 24 h, 48 h, and 72 h after tail vein injection, and then immediately dissected. Hearts, livers, spleens, lungs, and kidneys were collected, washed with PBS, and then EGFP fluorescence signals were detected and imaged using a VISQUE in vivo imaging system.

[0035] like Figure 14 and Figure 15 As shown, 48 h after administration, the fluorescence intensity of the NGS nanoparticle group in the liver was significantly higher than that of the GS-pDNA group, and the transfection efficiency was 1.5 times that of GS-pDNA, indicating that the NGS carrier with benzenesulfonamide group can effectively enhance the in vivo liver delivery efficiency.

[0036] 2. Hemolysis rate Approximately 1 mL of whole blood was collected from the orbital venous plexus of mice and immediately placed in an anticoagulant tube containing sodium heparin. The blood sample was centrifuged at 2000 rpm for 15 min at 4°C, and the supernatant fibrinogen was discarded. The red blood cells were then resuspended in physiological saline, and the centrifugation and washing process was repeated until the supernatant was clear. Finally, the red blood cell suspension was diluted to 3% (v / v) with physiological saline for later use. The 3% red blood cell suspension was mixed with GS-pDNA, NGS nanoparticles, NGS-PGA nanoparticles (10 kDa), and NGS-PGA nanoparticles (300 kDa) as experimental groups, respectively. Physiological saline was used as a negative control, and Triton 100X was used as a positive control. The mixtures were incubated at 37°C for 1 h, 5 h, and 24 h, respectively. After incubation at each time point, all samples were centrifuged at 2000 rpm for 15 min, the supernatant was carefully collected and photographed, and finally 100 μL of the supernatant from each group was taken into an ELISA reader and the corresponding absorbance was measured at a wavelength of 450 nm to calculate the hemolysis rate.

[0037] ; Where a is the hemolysis rate (%), OD1 is the absorbance of the negative control (physiological saline) group, OD2 is the absorbance of the positive control (Triton X-100) group, and OD3 is the absorbance of the experimental group.

[0038] like Figure 16 and Figure 17As shown, the hemolysis rate of GS-pDNA was 7.98% after 1 hour of treatment, with no significant hemolysis observed. However, significant hemolysis was observed after 5 hours, with a hemolysis rate of 12.29%, indicating the presence of hemolytic activity. After 24 hours of treatment, the hemolysis rate of GS-pDNA further increased to 16.61%. In contrast, after 24 hours of incubation with erythrocytes, the hemolysis rate of NGS nanoparticles was 8.86%, indicating that NGS nanoparticles significantly reduced the hemolysis rate of erythrocytes and possessed a certain degree of biocompatibility.

[0039] Experiment 5: Cell Viability The cytotoxicity of GS-pDNA, NGS nanoparticles, NGS-PGA nanoparticles (10 kDa), and NGS-PGA nanoparticles (300 kDa) was determined using the CCK-8 cell counting kit. A control group was established by adding DMEM instead of the complex, while a blank group was used without added cells. After incubating all experimental groups with cells for 24 h, the culture medium was aspirated, and 100 μL of CCK-8 reagent (CCK-8:DMEM (v / v) = 10:90) was added to each well. Incubation was continued for 1 h in the dark before termination. Absorbance values ​​were read at 450 nm using a microplate reader, and cell viability was calculated.

[0040] ; Where b represents cell viability (%), OD3 represents the absorbance of the experimental group, OD4 represents the absorbance of the blank group, and OD5 represents the absorbance of the negative control (DMEM) group.

[0041] like Figure 18 and Figure 19 As shown, the GS-pDNA group exhibited significant cytotoxicity after 24 hours of cell treatment, with a cell survival rate of only 62.8%, which was about 40% lower than that of the control group. In contrast, the cell survival rate increased to 72.3% after NGS nanoparticle treatment, indicating that NGS nanoparticles significantly improved the cytotoxicity of GS-pDNA.

[0042] Experimental Example 6: Cellular Uptake Pathway pDNA was labeled with Cy5 using the LabelIT® Nucleic Acid Labeling Kit (purchased from Mirus Bio, USA). Cells were prepared according to the manufacturer's instructions as follows: 20 μM chlorpromazine clathrin-mediated endocytosis inhibitor, 10 μM nystatin caveolin-mediated endocytosis inhibitor, and 10 μM cytochalasin D macropinocytosis inhibitor. A control group was prepared by pre-incubating cells in DMEM high-glucose medium (without FBS). Cells were pretreated with the above inhibitors at 37°C for 30 min, followed by the addition of Cy5-labeled NGS nanoparticles, NGS-PGA nanoparticles (10 kDa), and NGS-PGA nanoparticles (300 kDa) nanocomplexes, and incubated for 4 h. After washing twice with PBS, cell nuclei were stained with Hoechst 33342, and observation and recording were immediately performed under a confocal microscope.

[0043] like Figure 20 and Figure 21 As shown, the uptake of NGS nanoparticles did not change significantly under chlorpromazine pretreatment, but it was significantly reduced under nystatin pretreatment, down to 48.44% of the control group, suggesting that NGS may mainly enter cells through the pit-mediated endocytosis pathway.

[0044] Experimental Example 7: Cellular Uptake Efficiency AML-12 cells were loaded at 6 × 10 4 Cells were seeded at a density of [number] cells / well in 96-well plates using AML-12 culture medium and cultured overnight at 37°C and 5% CO2. Subsequently, nanoparticles containing Cy5-labeled GS-pDNA, NGS nanoparticles, NGS-PGA nanoparticles (10 kDa), and NGS-PGA nanoparticles (300 kDa) were added, and the cells were incubated at 37°C for 4 h. After incubation, the cells were washed twice with PBS, and the nuclei were stained with Hoechst 33342 at 37°C for 20 min. Immediate observation and recording were then performed under a confocal microscope.

[0045] like Figure 22 and Figure 23 As shown, the endocytosis efficiency of NGS nanoparticles was significantly higher than that of GS-pDNA, and its cellular uptake was 1.53 times that of GS-pDNA, indicating that NGS with benzenesulfonamide group modification helps to enhance the cellular uptake capacity of nanoparticles.

[0046] Experimental Example 8: Serum Stability NGS nanoparticles, NGS-PGA nanoparticles (10 kDa), and NGS-PGA nanoparticles (300 kDa) were added to 1.5 μL of 10% fetal bovine serum (FBS). The control group was treated with an equal volume of PBS instead of FBS. The binary / ternary complexes containing FBS were then incubated at 37°C for different times (0.5, 8, and 24 h). The particle size and dispersion index (PDI) of each sample were determined using a Malvern particle size distribution analyzer.

[0047] like Figure 24 and Figure 25 As shown, the particle size and PDI of NGS nanoparticles did not change significantly over time in PBS, but the particle size increased significantly from 161.7 nm to over 200 nm in 10% FBS. In contrast, the particle size and PDI of NGS-PGA nanoparticles (10 kDa) and NGS-PGA nanoparticles (30 kDa) did not change significantly in PBS and 10% FBS, indicating that the addition of γ-PGA can significantly improve the serum stability of NGS nanoparticles.

Claims

1. A method for preparing a nucleic acid delivery system, characterized in that, Includes the following steps: S1. Dissolve 2-(4-aminosulfonylphenyl)acetic acid in N,N′-carbonyldiimidazole and stir for 20-40 min. Then add 3,3′-iminobis(N,N-dimethylpropylamine), mix well, and reflux at 60-80℃ for 1-3 h to obtain the intermediate product. The molar ratio of 2-(4-aminosulfonylphenyl)acetic acid, N,N′-carbonyldiimidazole and 3,3′-iminobis(N,N-dimethylpropylamine) is 1-2:1-2:1-2. S2. The intermediate product and bromooctadecane are mixed and dissolved in a molar ratio of 0.5-1.5:2-3, and then refluxed at 90-110℃ for 70-74h to obtain the Gemini surfactant. S3. Mix the Gemini surfactant and the substance to be delivered at a mass ratio of 6-10:0.5-1.5, repeatedly blow and vortex for 5-8 seconds, and then incubate at room temperature for 0.5-1.5 hours to obtain NGS nanoparticles; the substance to be delivered is plasmid DNA, messenger RNA, small interfering RNA or antisense oligonucleotide.

2. The method for preparing the nucleic acid delivery system according to claim 1, characterized in that, The solvents used for dissolving in S1 and S2 are tetrahydrofuran or acetonitrile.

3. The method for preparing the nucleic acid delivery system according to claim 1, characterized in that, The molar ratio of 2-(4-aminosulfonylurea)acetic acid, N,N′-carbonyldiimidazole and 3,3′-iminobis(N,N-dimethylpropylamine) is 1:1:

1.

4. The method for preparing the nucleic acid delivery system according to claim 1, characterized in that, Also includes: The γ-polyglutamic acid solution and NGS nanoparticles were mixed at a ratio of 1.9-9.7 μL: 15-20 μg, and the mixture was repeatedly blown around 3-5 times. Then, it was incubated at room temperature for 30-60 min to obtain NGS-PGA nanoparticles.

5. The method for preparing the nucleic acid delivery system according to claim 4, characterized in that, The concentration of the γ-polyglutamic acid solution is 90-110 μg / mL.

6. A nucleic acid delivery system, characterized in that, It is prepared by the preparation method according to any one of claims 1-5.

7. The use of the nucleic acid delivery system according to claim 6 in the preparation of nucleic acid delivery drugs.

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