A gene delivery carrier, a nucleic acid nanoaggregate sensitive to high shear stress, and a preparation method and application thereof

Abstract

This invention discloses a gene delivery vector, highly shear stress-sensitive nucleic acid nanoaggregates, their preparation methods, and applications. The highly shear stress-sensitive gene delivery vector includes either polymer PLGA-PGEA or polymer PLGA-PGEA / TA. This invention utilizes ethanolamine and amino-modified tannic acid to functionalize polymer PLGA-PGEA, constructing polymer PLGA-PGEA and PLGA-PGEA / TA gene delivery vectors, exhibiting high cell transfection efficiency, low cytotoxicity, and suitable particle size, showing promise for application in gene therapy for cardiovascular diseases. The PLGA-PGEA / TA gene delivery vector of this invention uses PLGA as a hydrophobic core to stabilize nanoparticles, while the outer PGEA / TA layer improves its water solubility and can also complex genes, obtaining nucleic acid nanoaggregates with the required particle size. These not only safely accumulate in the stenotic region of coronary arteries but also exhibit sensitivity to high shear stress dispersion in the stenotic region, demonstrating significant application value and clinical translational potential in the targeted treatment of atherosclerosis.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a gene delivery vector, a nucleic acid nanoaggregate with high shear stress sensitivity, its preparation method, and its application. Background Technology

[0002] Gene therapy, as a cutting-edge biomedical technology, has shown increasingly broad and important application prospects in the diagnosis and treatment of various complex diseases, including inflammatory diseases, neurological diseases, and cardiovascular diseases. By precisely regulating gene expression or repairing defective genes, this strategy provides new intervention ideas and treatment possibilities for many refractory diseases. Although various gene delivery systems have been researched and reported, such as viral vectors, liposomes, and polymer nanoparticles, they still have many shortcomings in terms of safety, transfection efficiency, and targeting. This is especially true for the specific field of cardiovascular diseases, where the complexity of the vascular environment and the specificity of lesion sites place more stringent demands on the performance of gene delivery vectors. An ideal gene delivery vector should possess high cell transfection efficiency, low cytotoxicity, and an appropriate particle size to balance the relationship between delivery efficiency, in vivo circulation, and cellular uptake. Therefore, developing a safe and efficient gene delivery vector is of vital importance for promoting the clinical application of gene therapy in cardiovascular diseases.

[0003] Coronary artery stenosis caused by atherosclerosis is a common vascular disease in clinical practice, seriously affecting people's quality of life. The main clinical method for diagnosing atherosclerosis is computed tomography angiography (CTA). Based on CTA images, vascular hemodynamics are simulated, and the calculation results have certain reference value in clinical practice. After plaque formation, the blood flow characteristics within the coronary arteries change significantly. Due to the low spatial resolution of the coronary environment, and the fact that conventional medical angiography can only identify significant changes in the lumen diameter but not atherosclerosis itself, some studies have chosen to perform biomechanical modeling of the coronary arteries to supplement the limitations of medical imaging. For example, Sun et al. systematically studied the relationship between blood flow velocity, wall shear stress, and the formation of atherosclerotic plaques through simulation calculations and clinical comparisons; Qiao Aike et al. used computational fluid dynamics to numerically analyze the hemodynamics in tortuous arteries, finding that the wall shear stress on the inner wall of the bend is more pulsatile than that on the outer wall. Modeling coronary arteries at the level of biohemodynamics not only helps in the planning and evaluation of interventional procedures, but also enables a more comprehensive assessment of coronary artery pathology.

[0004] However, the complexity of coronary artery blood flow patterns after plaque formation involves not only changes in blood flow velocity and shear stress, but also turbulence and oscillations caused by residual fluid stress. These changes in physical conditions can lead to endothelial cell dysfunction. Furthermore, plaque sizes vary at different stages of atherosclerosis, resulting in different blood flow patterns. Therefore, a more in-depth analysis of flow velocity, shear stress, and blood flow patterns at each stage of atherosclerosis is needed to establish a more comprehensive coronary artery biomechanical model, providing new mechanisms and theoretical support for targeted clinical treatment.

[0005] Studies have found that once occlusive plaques caused by atherosclerosis form, they lead to high shear rates upstream and low flow rates downstream of the stenotic region. Upstream of the stenotic region, the reduced lumen radius due to stenosis increases blood flow shear rate and shear stress, peaking at the entrance of the stenosis. This can cause fatigue damage to the surface of the atherosclerotic plaque, physically destroying the fibrous cap covering the necrotic core and exposing the subendothelial layer to the blood. This, in turn, leads to platelet adhesion and aggregation, potentially triggering acute thrombosis. Downstream of the stenotic region, the high-velocity outflowing fluid forms a recirculation zone under the pressure differential. The prolonged blood flow stagnation and low shear rate create a region with low and turbulent blood flow. This can alter the arrangement of endothelial cells, making them more porous and increasing their permeability to macromolecules. Simultaneously, it upregulates pro-inflammatory molecules in endothelial cells, impairing endothelial function and causing the plaque to tend to extend further downstream. In response to this altered blood flow pattern and abnormal hemodynamics, some studies have proposed a strategy based on physical mechanisms rather than traditional molecular targeting. This strategy targets narrowed regions within blood vessels and utilizes the instantaneously increased shear stress to release drugs within these narrowed areas. Regarding this mechanism, research has found that nanoparticles, similar in size to platelets, are more prone to flowing along the vessel wall edges due to hydrodynamic forces in the bloodstream. Therefore, a key technology for achieving enrichment in narrowed coronary artery regions is the preparation of micron-sized aggregates (1-3 micrometers). However, these aggregates are relatively large, and current mainstream nanoscale delivery carriers are considered to pose risks such as vascular blockage if such large particles enter the blood vessels. One study proposed using relatively weak van der Waals forces to aggregate individual nanoparticles. When these aggregates are exposed to the high shear stress region caused by narrowing, they can overcome their adhesive forces and release individual nanoparticles, much like a clump of sand being dispersed into individual particles by hand. Once dispersed, these nanoparticles can deposit in the downstream recirculation region of the narrowed area, attaching to the plaque surface and further exerting a therapeutic effect. For example, Shen et al. constructed a drug delivery nanocarrier coated with a erythrocyte membrane-bound polyethyleneimine (PEI) cationic polymer. They utilized the electrostatic adsorption between the inherent negative charge on the erythrocyte membrane surface and the positive charge of the cationic polymer to form core-shell structured nanoparticles. High shear stress provided the driving force for the exfoliation of the erythrocyte membrane from the polymer, thus facilitating drug release. It is evident that the high shear dispersion capability of the aggregates significantly limits the risk of vascular occlusion, while weak intermolecular interactions provide support and assurance for the aggregation and shear dispersion of nanoparticles.

[0006] Current research on highly shear-sensitive delivery systems mainly focuses on drug delivery, with few reports on gene delivery. Therefore, based on the high blood flow shear stress in stenotic regions and the physical targeting properties of large-sized nanoparticles, developing a gene delivery system with both targeted enrichment capabilities and shear sensitivity in stenotic regions has significant scientific value and clinical translational potential for the targeted treatment of atherosclerosis. Summary of the Invention

[0007] To overcome the shortcomings of the existing technology, the present invention aims to provide a gene delivery vector with high cell transfection efficiency, low cytotoxicity and suitable particle size.

[0008] Another objective of this invention is to provide nucleic acid nanoaggregates with high shear stress sensitivity, which can not only be safely enriched in the narrowed area of ​​the coronary artery, but also have high sensitivity to the high shear stress in the narrowed area. They can be effectively dispersed under the action of high shear stress, thus achieving efficient and safe gene therapy.

[0009] This invention is achieved through the following technical solution: In a first aspect, the present invention provides a gene delivery vector with high shear stress sensitivity, comprising any one of polymer PLGA-PGEA or polymer PLGA-PGEA / TA; The structure of the polymer PLGA-PGEA is shown in Formula I below: Equation I, where x1, y1, and n1 represent the number of repetitions of the corresponding structural units, x1 = 40 - 216, y1 = 36 - 242, and n1 = 70 - 280; The structure of the polymer PLGA-PGEA / TA is shown in the following formula: Equation II, where x2, y2, n2, and m represent the number of repetitions of the corresponding structural units, x2 = 40 - 216, y2 = 36 - 242, n2 = 64 - 277, and m = 3 - 6.

[0010] Further, the number-average molecular weight of the polymer PLGA-PGEA is 24,000-70,000, preferably 30,000-55,000; the number-average molecular weight of the polymer PLGA-PGEA / TA is 24,000-80,000, preferably 35,000-60,000. The number-average molecular weight is determined by gel permeation chromatography (GPC).

[0011] This invention provides a method for preparing the polymer PLGA-PGEA and polymer PLGA-PGEA / TA, comprising the following steps: S1. Aminated tannic acid TA-NH2 was prepared by reacting tannic acid (TA) with ethylenediamine (ED) and N,N'-carbonyldiimidazole (CDI). S2. The macromolecular initiator PLGA-Br was prepared by reacting polylactic acid-glycolic acid copolymer (PLGA) with 2-bromo-2-methylpropionyl bromide (BIBB); S3. Using PLGA-Br as an initiator and glycidyl methacrylate (GMA) as a monomer, polymer PLGA-PGMA was prepared by atom transfer radical polymerization. S4. The polymer PLGA-PGEA is prepared by reacting PLGA-PGMA with ethanolamine via an amino-epoxy ring-opening reaction, or the polymer PLGA-PGEA / TA is prepared by reacting PLGA-PGMA with TA-NH2 and ethanolamine (EA) via an amino-epoxy ring-opening reaction.

[0012] Further, step S1 includes the following steps: dissolving tannic acid in dimethyl sulfoxide, purging the system with an inert gas to remove air, adding N,N'-carbonyldiimidazole, continuing to purge the system with an inert gas, reacting at 35-45℃ for 3-5 hours, then adding ethylenediamine, and continuing the reaction at 30-45℃ under an inert gas atmosphere for 20-30 hours; after the reaction is completed, dialyzing to remove unreacted small molecules, freeze-drying to prepare amino-modified tannic acid TA-NH2; wherein, the ratio of tannic acid to dimethyl sulfoxide is (50-60) mg: 1 mL; the mass ratio of N,N'-carbonyldiimidazole to tannic acid is 1: (5.0-5.5); and the mass ratio of ethylenediamine to tannic acid is 1: (1.3-1.5).

[0013] Further, step S2 includes the following steps: dissolving the polylactic acid-glycolic acid copolymer in N,N-dimethylformamide, adding triethylamine under ice bath conditions, and then adding 2-bromo-2-methylpropionyl bromide, reacting for 20-30 hours; after the reaction, precipitating with saturated sodium bicarbonate solution to obtain a powdered solid, washing and freeze-drying to prepare the macromolecular initiator PLGA-Br; wherein, the ratio of polylactic acid-glycolic acid copolymer to N,N-dimethylformamide is (120-150) mg: 1 mL; the mass ratio of triethylamine to polylactic acid-glycolic acid copolymer is 1: (3-4); and the mass ratio of 2-bromo-2-methylpropionyl bromide to polylactic acid-glycolic acid copolymer is 1: (1.3-1.5).

[0014] Preferably, the number-average molecular weight of the polylactic acid-glycolic acid copolymer is 9000-12000. More preferably, the molar ratio of the polymer monomers in the polylactic acid-glycolic acid copolymer is LA:GA = (0.3-3):1, and more preferably LA:GA = 1:1.

[0015] Further, step S3 includes the following steps: dissolving PLGA-Br in anhydrous dimethyl sulfoxide, purging the system with an inert gas to remove air, adding glycidyl methacrylate and a catalyst, reacting at 35-40℃ under an inert gas atmosphere for 1 h ± 10 min, and after the reaction is completed, precipitating, washing, and vacuum drying are performed to prepare the polymer PLGA-PGMA; wherein, the mass ratio of glycidyl methacrylate to PLGA-Br is (3-11):1; and the mass ratio of catalyst to PLGA-Br is 1:(3-4).

[0016] Preferably, the catalyst is 2,2'-bipyridine and cuprous bromide, and the mass ratio of 2,2'-bipyridine to cuprous bromide is (1.1-1.5):1.

[0017] The molecular weight of the polymer PLGA-PGMA can be controlled by adjusting the proportion of glycidyl methacrylate. Preferably, the mass ratio of glycidyl methacrylate to PLGA-Br is (5-6):1.

[0018] Further, step S4 includes the following steps: dissolving PLGA-PGMA in anhydrous dimethyl sulfoxide, purging the system with an inert gas to remove air, adding ethanolamine, and reacting at 35-45°C under an inert gas atmosphere for 10-15 hours; after the reaction is completed, dialyzing to remove unreacted small molecules, freeze-drying, and preparing the polymer PLGA-PGEA. Alternatively, step S4 includes the following steps: dissolving TA-NH2 in anhydrous dimethyl sulfoxide and sonicating in a water bath to obtain an aminotannic acid solution with a mass concentration of 9-45 mg / mL; dissolving PLGA-PGMA in anhydrous dimethyl sulfoxide, purging the system with an inert gas to remove air, adding the aminotannic acid solution, reacting at 35-45℃ under an inert gas atmosphere for 10-15 h, then adding ethanolamine and continuing the reaction for another 10-15 h; after the reaction is complete, dialyzing to remove unreacted small molecules, freeze-drying, and preparing the polymer PLGA-PGEA / TA; In step S4, the mass ratio of ethanolamine to PLGA-PGMA is (18-22):1.

[0019] In step S4, the mass ratio of PLGA-PGMA to TA-NH2 is (1-5.5):1. This invention can control the amount of tannic acid grafted onto the polymer PLGA-PGEA / TA by controlling the proportion of TA-NH2 used. Preferably, the mass ratio of PLGA-PGMA to TA-NH2 is (4-5):1.

[0020] Thirdly, the present invention provides a nucleic acid nanoaggregate, which is an aggregate formed by the aggregation of a complex of nucleic acid and the gene delivery vector; the gene delivery vector is a polymer PLGA-PGEA / TA.

[0021] Furthermore, the nitrogen-to-phosphorus ratio of the gene delivery vector to the nucleic acid is 20-30.

[0022] Further, the average particle size of the nucleic acid nanoaggregates is 0.3-1.5 micrometers; preferably, the average particle size of the nucleic acid nanoaggregates is 0.7-1.5 micrometers. The average particle size of the nucleic acid nanoaggregates can be 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4 or 1.5 micrometers, or specific values ​​between the above values.

[0023] Furthermore, the nucleic acid includes, but is not limited to, plasmids; the plasmids that can be selected include, but are not limited to, pDNA, siRNA, miRNA, etc.

[0024] Fourthly, the present invention provides a method for preparing the aforementioned nucleic acid nanoaggregates, comprising the following steps: Nucleic acid and gene delivery vector are mixed with a nitrogen-to-phosphorus ratio of 20-30 and diluted with water to obtain a mixed solution. A tannic acid solution with a mass concentration of 0.08-0.12 mg / mL is added, shaken well, and allowed to stand for 15-30 min to prepare nucleic acid nanoaggregates. The volume ratio of the mixed solution to the tannic acid solution is (0.5-1.8):1, preferably (1-1.5):1.

[0025] Fifthly, the present invention provides the application of the aforementioned gene delivery vector or nucleic acid nanoaggregate in the preparation of medicaments for treating cardiovascular diseases. Specifically, the cardiovascular diseases include, but are not limited to, coronary artery stenosis caused by atherosclerosis.

[0026] The present invention has the following beneficial effects: This invention utilizes ethanolamine and amino-modified tannic acid to functionalize the polymer PLGA-PGMA, constructing polymer PLGA-PGEA and PLGA-PGEA / TA gene delivery vectors, which have high cell transfection efficiency, low cytotoxicity, and suitable particle size, and are expected to be applied in gene therapy for cardiovascular diseases.

[0027] The PLGA-PGEA / TA gene delivery vector of the present invention uses PLGA as a hydrophobic core stabilized nanoparticle, and the outer PGEA / TA improves its water solubility while also complexing genes to obtain nucleic acid nanoaggregates with the required particle size. It can not only safely accumulate in the stenotic area of ​​the coronary artery, but also has the sensitivity to dispersion in response to high shear stress in the stenotic area. It has important application value and clinical translation potential in the targeted treatment of atherosclerosis. Attached Figure Description

[0028] Figure 1 The NMR characterization data of amino-modified tannic acid TA-NH2 in Example 1 are shown below. Figure 2 The NMR characterization data are for the macromolecular initiator PLGA-Br in Example 1; Figure 3 The NMR characterization data are for the polymer PLGA-PGMA-2 of Example 1; Figure 4 The NMR characterization data of polymer 30k PLGA1-PGEA / TA1 in Example 1 are as follows; Figure 5 The NMR characterization data of polymer 30k PLGA1-PGEA in Example 2 are as follows; Figure 6 The results of cell transfection and toxicity experiments in Example 3; Figure 7 The average particle size and particle size potential test results are for Example 3; Figure 8 The average particle size and particle size potential test results are for Examples 4-6; Figure 9 The results are from the CFD fluid model simulation and the maximum shear rate simulation under 80% blockage conditions in Example 7. Figure 10 This is a schematic diagram of the microfluidic chip design and in vitro fluid shearing experimental system in Example 7; Figure 11 The graph shows the particle size variation of nucleic acid nanoaggregates in Example 7 under a shear stress of 10.3 Pa. Figure 12 The results show the enrichment of nucleic acid nanoaggregates in narrow regions in Example 7; Figure 13 This is a scanning electron microscope image of the nucleic acid nanoaggregates 30k PLGA1-PGEA / TA1&TA in Example 7 before shearing. Figure 14 This is a scanning electron microscope image of the nucleic acid nanoaggregates 30k PLGA1-PGEA / TA1&TA in Example 7 after shearing. Detailed Implementation

[0029] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0030] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0031] Example 1 This embodiment provides a gene delivery vector, which is a polymer PLGA-PGEA / TA, and its structure is as follows: Where x=77, y=77, n=64-277, m=3-6. The preparation method is as follows: S1. Synthesis of amino-modified tannic acid TA-NH2:

[0032] 850 mg of tannic acid was weighed and dissolved in 15 mL of dimethyl sulfoxide. Nitrogen gas was bubbled through the solution for 5 min to purge the fumes. Then, 160 mg of N,N'-carbonyldiimidazole was added, and nitrogen gas was bubbled through the solution for another 5 min. The reaction was carried out at 40 °C for 4 h. Then, 629 mg of ethylenediamine was added, and the reaction was continued at 40 °C under a nitrogen atmosphere for 24 h. After the reaction was complete, the mixture was dialyzed through a 500 kDa dialysis bag for 3 days to remove unreacted small molecules. The solution was then freeze-dried to prepare aminoated tannic acid TA-NH2. Its NMR characterization data are as follows: Figure 1 As shown; S2. Synthesis of the macromolecular initiator PLGA-Br:

[0033] 100 mg of polylactic acid-glycolic acid copolymer (hydroxyl-terminated PLGA, LA:GA = 50:50, number average molecular weight 10000) was weighed and dissolved in 8 mL of N,N-dimethylformamide (DMF). 291 mg of triethylamine was added under ice bath conditions, followed by slow dropwise addition of 660 mg of 2-bromo-2-methylpropionyl bromide to the reaction system. The reaction was carried out under ice bath conditions for 24 h. After the reaction, a brownish-yellow powder was precipitated with saturated sodium bicarbonate solution. The powder was washed three times with saturated sodium bicarbonate solution and then freeze-dried to prepare the macromolecular initiator PLGA-Br. Its NMR characterization data are as follows: Figure 2 As shown; S3. Synthesis of polymer PLGA-PGMA:

[0034] 100 mg PLGA-Br was dissolved in 3 mL of anhydrous DMSO, and nitrogen gas was bubbled through the system for 5 min to purge air. Glycidyl methacrylate (the amounts are shown in Table 1), 31 mg 2,2'-bipyridine (2,2'-Bpy), and 28 mg cuprous bromide (CuBr) were added. The reaction was carried out at 37 °C under a nitrogen atmosphere for 1 h. After the reaction, the product was precipitated with anhydrous methanol and repeatedly washed and purified. Finally, the methanol was removed by vacuum drying to obtain a white powder solid, which was the target product polymer PLGA-PGMA. The NMR characterization data of polymer PLGA-PGMA-2 are as follows: Figure 3 As shown; Polymers PLGA-PGMA with different molecular weights were prepared according to the GMA monomer addition amounts in Table 1.

[0035] Table 1. GMA monomer addition amount and polymer molecular weight in the preparation of polymer PLGA-PGMA

[0036] S4. Synthesis of polymer PLGA-PGEA / TA:

[0037] Weigh TA-NH2 (the amount shown in Table 2) and dissolve it in 1 mL of anhydrous DMSO. Sonicate in a water bath for 1 min to obtain an aminotannic acid solution. Dissolve 50 mg PLGA-PGMA in anhydrous DMSO, purge the system with nitrogen for 5 min, add the aminotannic acid solution, and continue purging with nitrogen for another 5 min. React at 40 °C under an inert atmosphere for 12 h, then add 1012 mg ethanolamine and continue the reaction for another 12 h. After the reaction is complete, dialyze for 2 days to remove unreacted small molecules, freeze-dry, and prepare the polymer PLGA-PGEA / TA. The NMR characterization data of 30kJ PLGA1-PGEA / TA1 are as follows: Figure 4 As shown, its structural formula contains x=77, y=77, n=139, and m=3.

[0038] According to the TA-NH2 addition amount and the type of PLGA-PGMA used in Table 2, polymers PLGA-PGEA / TA with different molecular weights and different tannic acid grafting numbers were prepared.

[0039] Table 2. Type of PLGA-PGMA, amount of TA-NH2 added, polymer molecular weight, and number of tannic acid grafts in the preparation of polymer PLGA-PGEA / TA.

[0040] Example 2 This embodiment provides a gene delivery vector, which is a polymer PLGA-PGEA, and its structure is as follows: Where x=77, y=77, n=70-280. The preparation method differs from Example 1 only in that TA-NH2 is not added in step S4; the remaining steps are the same as in Example 1, preparing the ungrafted tannic acid polymer PLGA-PGEA:

[0041] According to the PLGA-PGMA type used in Table 3, polymers PLGA-PGEA of different molecular weights were prepared; among them, the NMR characterization data of 30k PLGA1-PGEA are as follows: Figure 5 As shown, in its structural formula, x=77, y=77, and n=142.

[0042] Table 3. Types of PLGA-PGMA and polymer molecular weights in the preparation of polymer PLGA-PGEA / TA

[0043] Example 3 The gene delivery vectors from Examples 1 and 2 were complexed with pDNA (luciferase reporter gene plasmid, extracted using Omega's endomycin-free mass extraction kit) under different nitrogen-to-phosphorus ratios (the nitrogen-to-phosphorus ratio was determined based on the effective cationic nitrogen in polymer PLGA-PGEA or polymer PLGA-PGEA / TA and the phosphorus in the phosphate group of the nucleic acid), and their cell transfection ability, relative cell viability, average particle size, and particle size potential were tested respectively.

[0044] 1. Cell transfection and toxicity experiments First, the transfection capability of the cationic nucleic acid complex was tested in HEK 293 cells. Using gold-labeled PEI (25 kDa) as a control, cells in the culture chamber were digested and counted, and approximately 100,000 cells were evenly seeded into 24-well plates. The plates were shaken well and incubated for 24 hours. When the cells reached approximately 70% confluence, PLGA-PGEA / pDNA and PLGA-PGEA / TA / pDNA were prepared into 6 groups with different N / P ratios (5, 10, 15, 20, 25, or 30). The amount of pDNA in each well was fixed at 1 μg. After co-incubating the nucleic acid complex with the cells for 4 hours, the culture medium was changed, and incubation continued for another 20 hours. The cells were gently washed with PBS, and then the PBS was aspirated. 70 μL of lysis buffer was added to each well to lyse the cells, and the plates were shaken for 2 hours and then placed in a -80°C freezer. The fluorescence intensity was then read using a cryoluminometer.

[0045] Using gold-labeled PEI as a control, cytotoxicity tests were first performed on HEK 293 cells. The MTT assay (thiazolyl blue) was used to evaluate the cytotoxicity of PLGA-PGEA / pDNA and PLGA-PGEA / TA / pDNA under different N / P conditions. Cells were first digested and counted, and then evenly seeded into 96-well plates at a density of 10,000 cells per well. The plates were shaken and incubated for 24 hours. PLGA-PGEA / pDNA and PLGA-PGEA / TA / pDNA were configured into six groups with different N / P ratios (5, 10, 15, 20, 25, or 30). The amount of pDNA in each well was fixed at 0.25 μg. After static complexation for 30 min, the pDNA was diluted to 1 mL with culture medium. Six parallel wells were set up for each group, and the cells were incubated for 4 hours. The culture medium was then changed, and the cells were incubated for another 20 hours. Weigh 5 mg MTT and dissolve it in 1 mL PBS buffer. Add 9 mL of serum-free high-glucose medium to dilute the MTT concentration to 0.5 mg / mL. Perform the entire process in the dark. Slowly aspirate the old culture medium and add 100 μL of MTT solution to each well. Incubate for 4 h. After incubation, remove the old MTT solution, add 100 μL of LDMSO to each well, and vortex to mix. Read the UV absorbance at 490 nm using a microplate reader. Calculate cell viability using the following formula: In the formula, the dead group is the group of cells cultured in water, and the live group is the group of cells cultured in culture medium.

[0046] 2. Average particle size and particle size potential test Each sample was set to contain 5 μg of nucleic acid. The complex was prepared into 6 groups of samples according to different N / P ratios (5, 10, 15, 20, 25 or 30), and diluted with ultrapure water to 800 μL. After standing for 30 min, the samples were tested using a dynamic light scattering (DLS) instrument.

[0047] Test results are as follows Figure 6 , Figure 7 As shown.

[0048] Depend on Figure 6The results show that the gene delivery vector of the present invention exhibits high cell transfection efficiency and low cytotoxicity. For PLGA-PGEA without tannic acid grafting, the optimal nitrogen-to-phosphorus ratio of 30k PLGA1-PGEA to nucleic acid complexation is 20, which also shows excellent high transfection efficiency and low toxicity. After grafting tannic acid, the transfection ability is inversely proportional to the number of tannic acids grafted. After grafting 6 tannic acids, the transfection ability decreases to some extent compared to grafting 3 tannic acids. For polymers grafted with 3 tannic acids, the optimal nitrogen-to-phosphorus ratio of 50k PLGA1-PGEA / TA1 is 20, which also shows excellent high transfection efficiency and low toxicity. For materials grafted with 3 tannic acids, the optimal nitrogen-to-phosphorus ratio of 30k PLGA1-PGEA / TA1 is 30, which has the highest cell transfection efficiency and low toxicity.

[0049] Depend on Figure 7 The results show that after the gene delivery vector of the present invention complexes nucleic acids, the particle size is around 200 nm and the potential is around 30 mV.

[0050] Example 4 Preparation of nucleic acid nanoaggregates: Each sample contained 5 μg of nucleic acid pDNA. 30kJ of polymer PLGA1-PGEA / TA1 gene delivery vector was added to each sample with a nitrogen-to-phosphorus ratio (N / P) of 30. The mixture was diluted with water to 480 μL. Subsequently, 320 μL, 400 μL, and 480 μL of tannic acid solution of different mass concentrations (0.1 mg / mL, 0.5 mg / mL, or 1 mg / mL) were added to each group of samples. After shaking and standing for 30 min, nucleic acid nanoaggregates were prepared.

[0051] Example 5 Preparation of nucleic acid nanoaggregates: Each sample contained 5 μg of nucleic acid pDNA. 50kJ of polymer PLGA1-PGEA / TA1 gene delivery vector was added to each sample with a nitrogen-to-phosphorus ratio (N / P) of 20. The mixture was diluted with water to 480 μL. Subsequently, 320 μL, 400 μL, and 480 μL of tannic acid solution of different mass concentrations (0.1 mg / mL, 0.5 mg / mL, or 1 mg / mL) were added to each group of samples. After shaking and standing for 30 min, nucleic acid nanoaggregates were prepared.

[0052] Example 6 The only difference between this embodiment and Example 5 is that the gene delivery vector is polymer 30k PLGA1-PGEA; the other steps are the same as in Example 5.

[0053] The test results of the average particle size and particle size potential of the nucleic acid nanoaggregates in Examples 4-6 are as follows: Figure 8 As shown.

[0054] Depend onFigure 8 The results show that the polymer PLGA1-PGEA / TA gene delivery vector of the present invention, by selecting natural polyphenol tannic acid, utilizes the multi-hydroxyl structure of the tannic acid monomer itself to form a hydrogen bond cross-linking network with the tannic acid grafted onto the material, which can effectively form aggregates. Polymer 30k PLGA1-PGEA / TA1 exhibits excellent aggregation effect under the condition of adding 320μL of 0.1 mg / mL tannic acid solution, effectively forming nucleic acid nanoaggregates of approximately 1μm; polymer 50k PLGA1-PGEA / TA1, due to its larger molecular weight and relatively lower tannic acid grafting ratio, forms smaller aggregates; for polymer 30k PLGA1-PGEA without tannic acid grafting, no aggregates can be formed under any tannic acid concentration or volume.

[0055] Example 7 This embodiment designs microfluidic channels with different blockage areas, uses peristaltic pumps and biomimetic pipes to build a fluid circulation shear system, and combines computational fluid dynamics (CFD) simulation to calculate the fluid shear rate with different flow rates and different blockage areas, further verifying the responsiveness of nucleic acid nanoaggregates to different shear stresses.

[0056] 1. Combining CFD and microfluidic chip to simulate hemodynamics at atherosclerotic plaques and build a fluid shearing system: Since the obstruction area of ​​the vascular lumen caused by the plaque varies depending on the stage of atherosclerosis, five sets of microfluidic channels with obstruction areas ranging from 0% to 80% were designed according to the size of the plaque to simulate the vascular environment under plaque obstruction; and based on the Comsol fluid simulation software, a pipe model with a diameter of 1 mm and different obstruction area sizes in the middle was built, with flow rates of 2 mL / min, 3 mL / min, and 4 mL / min respectively, water was selected as the fluid, boundary conditions were set, and a mesh was built before simulation.

[0057] 2. Construction of the in vitro fluid shearing system: Five sets of microfluidic channels with blockage areas ranging from 0% to 80% were designed. External peristaltic pumps were connected, and flow rates were set to 2 mL / min, 3 mL / min, and 4 mL / min, respectively. After connecting external biomimetic tubing, the prepared solution was drawn into the tubing, and cyclic shearing experiments were conducted for 10 min, 30 min, and 60 min, respectively. In the circulation system, only the microfluidic channels had blockages of varying areas; the remaining tubing maintained a uniform diameter.

[0058] The results of CFD fluid model simulation and maximum shear rate simulation under 80% choke conditions are as follows: Figure 9 As shown.

[0059] Simulation conditions: Flow rate: 3.3 mL / min; Liquid: water; Viscosity: 1.0 × 10⁻⁶ -3 Pa·s.

[0060] Calculation formula: Shear stress (τ) = Shear rate (γ) × Liquid viscosity (η).

[0061] Settlement results: Under the simulated conditions of 80% pore blockage, the maximum shear stress is 10.3 Pa.

[0062] Design of microfluidic chips and in vitro fluid shearing experimental systems, such as Figure 10 As shown.

[0063] Under a shear stress of 10.3 Pa, the particle size change of nucleic acid nanoaggregates after 30 min of shear testing is as follows: Figure 11 As shown.

[0064] To verify that the cleavage of nucleic acid nanoparticle aggregates facilitates endocytosis, pEGFP plasmid was used to observe the expression of green fluorescent protein in cells. HEK 293 cells were used. Before cell seeding, 50 μg / mL and 70 μL of fibronectin were seeded in normal and 80% blocked microfluidic channels, respectively. After coagulation for 1 h, the cells were digested and counted, and evenly seeded in each microfluidic channel at a rate of 2 weeks. The cells were then incubated for 24 h. Using the optimal N / P ratio and 0.1 mg / mL tannic acid solution, 50 μL of sample solutions containing 1 μg pEGFP in 30kJ PLGA1-PGEA / TA1 and 30kJ PLGA1-PGEA were prepared according to the preparation process in Examples 4-6, and allowed to stand for 30 min. The sample solutions were then neutralized with 1 mL of culture medium and circulated continuously at a flow rate of 3.3 mL / min for 30 min. The cells were then incubated for another 24 h, and the expression of green fluorescent protein was observed using a fluorescence microscope. The enrichment characteristics of the narrow area are as follows: Figure 12 As shown.

[0065] Scanning electron microscopy images of nucleic acid nanoaggregates 30k PLGA1-PGEA / TA1&TA before and after cleavage are shown below. Figure 13 , Figure 14 As shown.

[0066] The results show that, compared with PLGA-PGEA&TA, PLGA-PGEA / TA&TA nucleic acid nanoaggregates can respond to high shear stress dispersion, and the particle size of the aggregates recovers to about 300 nm after shear dispersion.

[0067] Depend on Figure 12The results show that the 30k PLGA1-PGEA polymer exhibits high uptake and expression at various locations in cells due to its strong transfection capability. The uptake and expression of 30k PLGA1-PGEA / TA1&TA at the narrow entry point and the recirculation region after narrowing are significantly higher than those of 30k PLGA1-PGEA, while expression at other locations is lower. Overall, the uptake and expression of 50k PLGA1-PGEA / TA1&TA are relatively low. This demonstrates that the gene delivery vector PLGA-PGEA / TA of this invention can form nucleic acid nanoaggregates through a hydrogen-bonded cross-linking network, exhibiting high shear stress-responsive dispersion at narrow locations and effective enrichment at the narrow entry point and the recirculation region after narrowing.

[0068] The above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A gene delivery vector, characterized in that, Including either polymer PLGA-PGEA or polymer PLGA-PGEA / TA; The structure of the polymer PLGA-PGEA is shown in Formula I below: Equation I, where x1, y1, and n1 represent the number of repetitions of the corresponding structural units, x1 = 40 - 216, y1 = 36 - 242, and n1 = 70 - 280; The structure of the polymer PLGA-PGEA / TA is shown in Formula II below: Equation II, where x2, y2, n2, and m represent the number of repetitions of the corresponding structural units, x2 = 40 - 216, y2 = 36 - 242, n2 = 64 - 277, and m = 3 - 6.

2. The gene delivery vector according to claim 1, characterized in that, The number average molecular weight of the polymer PLGA-PGEA is 24,000-70,000; the number average molecular weight of the polymer PLGA-PGEA / TA is 24,000-80,000.

3. The gene delivery vector according to claim 1, characterized in that, The preparation methods of the polymers PLGA-PGEA and PLGA-PGEA / TA include the following steps: S1. Aminated tannic acid TA-NH2 was prepared by reacting tannic acid with ethylenediamine and N,N'-carbonyldiimidazole. S2. The macromolecular initiator PLGA-Br was prepared by reacting polylactic acid-glycolic acid copolymer with 2-bromo-2-methylpropionyl bromide; S3. Using PLGA-Br as an initiator and glycidyl methacrylate as a monomer, polymer PLGA-PGMA was prepared by atom transfer radical polymerization. S4. The polymer PLGA-PGEA is prepared by reacting PLGA-PGMA with ethanolamine via an amino-epoxy ring-opening reaction, or the polymer PLGA-PGEA / TA is prepared by reacting PLGA-PGMA with TA-NH2 and ethanolamine via an amino-epoxy ring-opening reaction.

4. The gene delivery vector according to claim 3, characterized in that, In the preparation methods of the polymers PLGA-PGEA and PLGA-PGEA / TA, Step S1 includes the following steps: dissolving tannic acid in dimethyl sulfoxide, purging the system with an inert gas to remove air, adding N,N'-carbonyldiimidazole, continuing to purge the system with an inert gas, reacting at 35-45℃ for 3-5 hours, then adding ethylenediamine, and continuing the reaction at 30-45℃ under an inert gas atmosphere for 20-30 hours; after the reaction is complete, dialysis is performed to remove unreacted small molecules, followed by freeze drying to prepare amino-modified tannic acid TA-NH2; wherein, the ratio of tannic acid to dimethyl sulfoxide is (50-60) mg: 1 mL; the mass ratio of N,N'-carbonyldiimidazole to tannic acid is 1: (5.0-5.5); and the mass ratio of ethylenediamine to tannic acid is 1: (1.3-1.5). Step S2 includes the following steps: dissolving polylactic acid-glycolic acid copolymer in N,N-dimethylformamide, adding triethylamine under ice bath conditions, and then adding 2-bromo-2-methylpropionyl bromide, reacting for 20-30 hours; after the reaction, precipitating with saturated sodium bicarbonate solution to obtain a powdered solid, washing and freeze-drying to prepare the macromolecular initiator PLGA-Br; wherein, the ratio of polylactic acid-glycolic acid copolymer to N,N-dimethylformamide is (120-150) mg: 1 mL; the mass ratio of triethylamine to polylactic acid-glycolic acid copolymer is 1: (3-4); and the mass ratio of 2-bromo-2-methylpropionyl bromide to polylactic acid-glycolic acid copolymer is 1: (1.3-1.5); Step S3 includes the following steps: dissolving PLGA-Br in anhydrous dimethyl sulfoxide, purging the system with an inert gas to remove air, adding glycidyl methacrylate and a catalyst, reacting at 35-40℃ under an inert gas atmosphere for 1 h ± 10 min, and after the reaction is completed, precipitating, washing, and vacuum drying are performed to prepare the polymer PLGA-PGMA; wherein, the mass ratio of glycidyl methacrylate to PLGA-Br is (3-11):1; and the mass ratio of catalyst to PLGA-Br is 1:(3-4).

5. The gene delivery vector according to claim 3, characterized in that, In the preparation methods of the polymers PLGA-PGEA and PLGA-PGEA / TA, Step S4 includes the following steps: dissolving PLGA-PGMA in anhydrous dimethyl sulfoxide, purging the system with an inert gas to remove air, adding ethanolamine, and reacting at 35-45℃ under an inert gas atmosphere for 10-15 hours; after the reaction is completed, dialyzing to remove unreacted small molecules, freeze-drying, and preparing the polymer PLGA-PGEA. Alternatively, step S4 includes the following steps: dissolving TA-NH2 in anhydrous dimethyl sulfoxide and sonicating in a water bath to obtain an aminotannic acid solution with a mass concentration of 9-45 mg / mL; dissolving PLGA-PGMA in anhydrous dimethyl sulfoxide, purging the system with an inert gas to remove air, adding the aminotannic acid solution, reacting at 35-45℃ under an inert gas atmosphere for 10-15 h, then adding ethanolamine and continuing the reaction for another 10-15 h; after the reaction is complete, dialyzing to remove unreacted small molecules, freeze-drying, and preparing the polymer PLGA-PGEA / TA; The mass ratio of ethanolamine to PLGA-PGMA is (18-22):1; the mass ratio of PLGA-PGMA to TA-NH2 is (1-5.5):

1.

6. The gene delivery vector according to claim 4, characterized in that, In the preparation methods of the polymers PLGA-PGEA and PLGA-PGEA / TA, The catalyst in step S3 is 2,2'-bipyridine and cuprous bromide, wherein the mass ratio of 2,2'-bipyridine to cuprous bromide is (1.1-1.5):1; In step S3, the mass ratio of glycidyl methacrylate to PLGA-Br is (5-6):1; the mass ratio of catalyst to glycidyl methacrylate is 1:(16-20).

7. A nucleic acid nanoaggregate with high shear stress sensitivity, characterized in that, It is an aggregate formed by the aggregation of a nucleic acid and the gene delivery vector of any one of claims 1-6; the gene delivery vector is a polymer PLGA-PGEA / TA.

8. The nucleic acid nanoaggregate according to claim 7, characterized in that, The average particle size of the nucleic acid nanoaggregates is 0.3-1.5 micrometers; preferably, the average particle size of the nucleic acid nanoaggregates is 0.7-1.5 micrometers.

9. The method for preparing nucleic acid nanoaggregates according to any one of claims 7-8, characterized in that, Includes the following steps: Nucleic acid and gene delivery vector were mixed with a nitrogen-to-phosphorus ratio of 20-30 and diluted with water to obtain a mixed solution. A tannic acid solution with a mass concentration of 0.08-0.12 mg / mL was added, and the mixture was shaken and allowed to stand for 15-30 min to prepare nucleic acid nanoaggregates. The volume ratio of the mixed solution to the tannic acid solution was (0.5-1.8):

1.

10. The use of the gene delivery vector according to any one of claims 1-6 or the nucleic acid nanoaggregate according to any one of claims 7-8 in the preparation of a medicament for treating cardiovascular diseases.

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