Ionizable amino modified double-mesoporous silicon dioxide nano-particles as well as preparation method and application thereof

Abstract

The invention discloses double-mesoporous silica nanoparticles for modifying ionizable amino and a preparation method and application of the double-mesoporous silica nanoparticles. The core technical process of the double-mesoporous silica nanoparticles comprises the following steps: firstly, synthesizing mesoporous silica through a double-template method of a block copolymer and a surfactant; then grafting ionizable amino to the surface of the mesoporous silica through a silane coupling agent; and finally, RNA is loaded on the modified mesoporous silica by electrostatic adsorption, and efficient tumor treatment is finally realized. The invention also discloses an application of the silicon dioxide nanoparticles in RNA delivery. The silicon dioxide nanoparticles prepared according to the method disclosed by the invention have high RNA loading efficiency and cell uptake efficiency. The double-mesoporous silica nanoparticles modified with ionizable amino groups have the advantages of large drug loading capacity, high cell uptake rate, high biocompatibility, simplicity in synthesis, mild reaction conditions, convenience in cutting and the like, and have a good application prospect.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and more specifically to a modified ionizable amine-based dual-mesoporous silica nanoparticle, its preparation method, and its application. Background Technology

[0002] Currently, RNA delivery vectors are mainly divided into two categories: viral vectors and non-viral vectors. Viral vectors (such as adeno-associated virus (AAV) and lentiviruses) have high transfection efficiency, but they also have problems such as strong immunogenicity, potential carcinogenicity, limited load capacity, and complex manufacturing processes, making their clinical application relatively difficult. Among non-viral vectors, lipid nanoparticles are currently the mainstream vector for mRNA vaccines (such as Pfizer / BioNTech and Moderna's COVID-19 vaccines). They can form a stable core-shell structure with RNA, effectively preventing degradation by RNases, and are currently the only RNA delivery system approved for clinical use. Polymer vectors load RNA through charge interactions and can achieve customized delivery to specific cells through chemical coupling of targeting groups. They are also inexpensive and easy to industrialize.

[0003] However, lipid nanoparticles have drawbacks such as excessive liver targeting, short in vivo circulation time, and susceptibility to inflammation. Polymer carriers, on the other hand, are prone to cytotoxicity and inflammation. Therefore, it is necessary to design RNA delivery carriers to achieve high loading efficiency and cellular uptake efficiency. Furthermore, the biocompatibility of these carriers also needs further improvement.

[0004] Therefore, it is necessary to provide a novel RNA delivery vector with excellent biocompatibility, high RNA loading efficiency and cellular uptake efficiency, and the ability to effectively avoid defects such as pathogenicity, cytotoxicity and inflammatory response, in order to meet the demand for efficient RNA delivery in the treatment of diseases such as tumors. Summary of the Invention

[0005] The purpose of this invention is to provide a modified ionizable amino group dual mesoporous silica nanoparticle, its preparation method and application, thereby solving the problem that RNA delivery vectors in the prior art cannot achieve both high delivery and high biocompatibility.

[0006] To solve the above problems, the present invention adopts the following technical solution:

[0007] According to a first aspect of the present invention, a method for preparing bimesoporous silica nanoparticles modified with ionizable amine groups is provided. The method comprises the following steps: S1, dissolving a polystyrene-polyacrylic acid block copolymer and a polystyrene homopolymer in tetrahydrofuran to obtain a mixture 1; S2, dissolving a surfactant in ultrapure water and adding ammonia to obtain a mixture 2; S3, adding a silicon source to an ethanol solution to obtain a mixture 3; S4, stirring the mixture 1 until homogeneous and then pouring it into the mixture 2 to obtain a mixture 4; S5, pouring the mixture 4 into the mixture 3 to obtain... S5: Mixture 5 is stirred and allowed to stand; S7: Mesoporous silica nanoparticles are collected by centrifugation; S8: The collected mesoporous silica nanoparticles are calcined in a muffle furnace at 500-600℃ to remove polymers and surfactants; S9: The collected mesoporous silica is dispersed in toluene, and a silane coupling agent with ionizable amino groups is added to obtain mixture 6; S10: Mixture 6 is heated, stirred, and protected under a nitrogen atmosphere; S11: Mesoporous silica nanoparticles with surface-modified ionizable amino groups are collected by centrifugation. It should be understood that surfactants are the source of small mesopores in the mesoporous silica nanoparticles, while polystyrene-polyacrylic acid block copolymers and polystyrene homopolymers are the source of large mesopores. Their mass ratio affects the morphology and pore size of the nanoparticles.

[0008] Preferably, in step S1, the polystyrene-polyacrylic acid block copolymer has a polystyrene moiety with a molecular weight range of 5200 Da-41600 Da and a polyacrylic acid moiety with a molecular weight range of 720 Da-1440 Da. More preferably, the polystyrene moiety has a molecular weight of 15600 Da and the polyacrylic acid moiety has a molecular weight of 1080 Da. Block copolymers outside the preferred ranges will result in the inability to successfully prepare mesoporous silica nanoparticles.

[0009] Preferably, in step S1, the molecular weight of the polystyrene homopolymer ranges from 5200 Da to 41600 Da. More preferably, the molecular weight of the polystyrene homopolymer is 15600 Da. It should be understood that the molecular weight of the polystyrene homopolymer should be as close as possible to the molecular weight of the polystyrene portion in the polystyrene-polyacrylic acid block copolymer. If the difference is too large or the homopolymer exceeds the preferred range, it will damage the mesoporous structure of the mesoporous silica.

[0010] Preferably, in step S2, the surfactant is selected from one or more of dodecyltrimethylammonium bromide, hexadecyltrimethylammonium bromide, and octadecyltrimethylammonium bromide. In a preferred embodiment, the surfactant is octadecyltrimethylammonium bromide.

[0011] Preferably, the mass ratio of polystyrene-polyacrylic acid block copolymer, polystyrene homopolymer, and surfactant is (0.25-1):(0.1-1):(0.25-1). It should be understood that different raw material ratios will have a certain impact on the morphology and pore size of the nanoparticles. When the mass ratio of polystyrene-polyacrylic acid block copolymer to surfactant is close to 1:1, mesoporous silica tends to form an ellipsoidal morphology, while when the mass ratio is 0.25:1, a spherical morphology is more likely to form. The content of polystyrene homopolymer affects the pore size of silica. When the content is within the preferred range, the pore size of silica increases with the increase of polystyrene homopolymer content; when the content is higher than the preferred range, the effect on pore size is no longer significant. In a preferred embodiment, the mass ratio of polystyrene-polyacrylic acid block copolymer, polystyrene homopolymer, and surfactant is 0.25:0.4:1.

[0012] Preferably, the silicon source in step S3 is selected from one or more of tetraethyl orthosilicate, methyl orthosilicate, and sodium silicate. In a preferred embodiment, the silicon source is tetraethyl orthosilicate.

[0013] Preferably, in step S6, the stirring rate is 500-1300 rpm, the stirring time is 10-20 minutes, and the settling time is 10-24 hours. It should be understood that excessively high or low stirring rates can affect the uniformity of the mesoporous silica. In a preferred embodiment, the stirring rate is 600 rpm, the stirring time is 10 minutes, and the settling time is 18 hours.

[0014] Preferably, in step S8, the centrifuged and collected dual-mesoporous silica nanoparticles are placed in a muffle furnace and calcined at a rate of 1°C / min to 500-600°C, and held at that temperature for 360 min to remove polymers and surfactants. In a preferred embodiment, the centrifuged and collected dual-mesoporous silica nanoparticles are placed in a muffle furnace and calcined at a rate of 1°C / min to 550°C, and held at that temperature for 360 min to remove polymers and surfactants.

[0015] It should be understood that silane coupling agents with ionizable amino groups are a class of functional compounds whose molecular structure simultaneously contains the basic skeleton of a silane coupling agent (silicon atoms connecting a hydrolyzable group and an organic chain) and an ionizable amino functional group. Their core characteristic is that the typical structure of the silane moiety is YR-SiX3 (where X is a hydrolyzable group, such as methoxy or ethoxy; R is an alkylene group; and Y is an organic functional group), and an ionizable amino group (such as secondary amine −NHR, tertiary amine −NR2) is introduced into Y or R. Preferably, in step S8, the ionizable amino group is selected from: diethylamine, dimethylamine, piperidine, piperazine, N-methylpiperazine, imidazole, etc. In a preferred embodiment, the ionizable group is a diethylamine group.

[0016] According to a second aspect of the present invention, a modified ionizable amino group-modified dual-mesoporous silica nanoparticle is provided, prepared by the above-described method. The nanoparticle has a dual-mesoporous structure, with small mesopores having a diameter of 1-5 nm and large mesopores having a diameter of 10-50 nm. The nanoparticle possesses high RNA loading efficiency and cellular uptake efficiency.

[0017] According to the present invention, the ionizable amino groups in the nanoparticles load RNA through electrostatic adsorption, and achieve high cellular uptake efficiency through affinity with the cell membrane. The larger mesopores (10-50 nm) enable large-volume and efficient loading.

[0018] According to a third aspect of the present invention, there is an application of bimesoporous silica nanoparticles modified with ionizable amine groups in RNA delivery.

[0019] The present invention provides a method for preparing bimesoporous silica nanoparticles modified with ionizable amino groups, the core technical process of which is as follows: Figure 1As shown, firstly, mesoporous silica is synthesized using a dual-template method with block copolymers and surfactants. This step is the basic structural preparation of the carrier. The block copolymer (e.g., polystyrene-polyacrylic acid) and surfactant (e.g., octadecyltrimethylammonium bromide) serve as templates, guiding the formation of a dual-mesoporous structure (small mesopores ~2 nm, large mesopores 10-50 nm). Sufficient pore space is the structural basis for subsequent RNA loading. Secondly, ionizable amino groups (e.g., diethylamine groups) are grafted onto the surface of the mesoporous silica using a silane coupling agent. This is a key modification for the carrier to achieve its function. The ionizable amino groups protonate (become positively charged) under physiological conditions, enabling them to adsorb negatively charged RNA via electrostatic interactions and change their charge in the acidic environment of lysosomes, facilitating RNA release from the carrier and improving intracellular delivery efficiency. Finally, RNA (e.g., siRNA) is loaded onto the modified mesoporous silica using electrostatic adsorption, ultimately achieving highly efficient tumor treatment. The negatively charged RNA binds to the positively charged amine groups on the carrier surface via electrostatic interactions. After the loaded carrier is taken up by tumor cells, the RNA knocks down the target gene (such as AURKB), inhibiting tumor cell proliferation and achieving a therapeutic effect.

[0020] It should be understood that while existing technologies can successfully prepare dual-mesoporous silica nanoparticles with similar morphologies, they have never been modified with ionizable amino groups on their surfaces, nor have they been used for RNA delivery to achieve efficient tumor treatment. Compared to lipid nanoparticles, traditional methods of modifying inorganic nanoparticles with amino groups to load RNA have lower delivery efficiency. Therefore, existing RNA delivery technologies have not focused on carriers based on mesoporous silica. This invention uses surface-modified ionizable amino groups instead of amino groups, improving the RNA delivery efficiency of mesoporous silica while maintaining the high biocompatibility of the carrier.

[0021] The key inventive point of this invention lies in the fact that, utilizing the high biocompatibility of silica nanoparticles and the dynamic change in the charge state of ionizable amino groups with environmental pH as explained by the proton sponge effect, ionizable amino groups are modified onto bi-mesoporous silica nanoparticles for the first time. The modified bi-mesoporous silica nanoparticles with ionizable amino groups are mostly unprotonated in blood (pH 7.4), thus avoiding recognition and clearance by macrophages, while simultaneously reducing electrostatic repulsion with normal cell membranes and increasing the probability of nanoparticle uptake by cells. Secondly, the silica nanoparticles exhibit high safety, degrading into non-toxic silicic acid in lysosomes without long-term toxic accumulation. Therefore, the bi-mesoporous silica nanoparticles modified with ionizable amino groups prepared according to this invention further enhance the biocompatibility of the carrier compared to lipid nanoparticles.

[0022] Current RNA delivery vectors include viral vectors, lipid nanoparticles, and polymer vectors. Compared with existing technologies, the modified ionizable amino-group-modified bimesoporous silica nanoparticles, their preparation method, and applications provided by this invention have the following significant advantages:

[0023] 1) By using silane with ionizable amino groups to form an ionizable layer on the surface of double mesoporous silica nanoparticles, the unprotonated properties in the blood can prevent them from being recognized and cleared by macrophages. At the same time, it can reduce the electrostatic repulsion with normal cell membranes, increase the probability of nanoparticles being taken up by cells, and has good RNA delivery efficiency.

[0024] 2) The method for preparing modified mesoporous silica nanoparticles with ionizable amino groups according to the present invention utilizes a dealcoholization condensation reaction to modify the surface of the mesoporous silica nanoparticles with ionizable amino groups. The preparation process is simple, the reaction conditions are mild, the reproducibility is good, and it is convenient for large-scale production.

[0025] 3) The silica nanoparticles prepared by the present invention have high safety, and the degradation products are non-toxic silicic acid. Therefore, the nanoparticles prepared by the present invention have higher biocompatibility.

[0026] In summary, the present invention provides modified ionizable amino group-modified bi-mesoporous silica nanoparticles. These nanoparticles exhibit a dynamic change in charge state with environmental pH, which prevents them from being recognized and cleared by macrophages. Simultaneously, they reduce electrostatic repulsion with normal cell membranes, increasing the probability of cellular uptake. Furthermore, these nanoparticles possess good biocompatibility, degrading into non-toxic silicic acid within lysosomes without long-term toxic accumulation. The modified ionizable amino group-modified bi-mesoporous silica nanoparticles prepared according to the method of the present invention simultaneously possess advantages such as high loading efficiency, high cellular uptake efficiency, good biocompatibility, simple synthesis conditions, and mild reaction conditions, demonstrating promising application prospects. Attached Figure Description

[0027] Figure 1 This is a flowchart of a method for preparing ionizable mesoporous silica according to the present invention;

[0028] Figure 2 A transmission electron microscope (TEM) image of the diethylamine mesoporous silica from Example 1 is shown.

[0029] Figure 3 A scanning electron microscope image of the diethylamine mesoporous silica in Example 1 is shown;

[0030] Figure 4 The pore size distribution diagram of diethylamine mesoporous silica DFT in Example 1 is shown;

[0031] Figure 5The EDS elemental analysis diagram of the diethylamine mesoporous silica in Example 1 is shown;

[0032] Figure 6 This is a statistical chart showing the toxicity of diethylamine mesoporous silica to normal cells (293T cells) in Example 1;

[0033] Figure 7 This is an agarose gel electrophoresis image of siRNA loaded on diethylamine mesoporous silica in Example 1;

[0034] Figure 8 This is a statistical graph of phagocytosis of diethylamine mesoporous silica in Example 1 by mouse colon cancer cells;

[0035] Figure 9 This is a Western blot analysis of AURKB knockout in mouse colon cancer cells using diethylamine mesoporous silica loaded with siAURKB as described in Example 1.

[0036] Figure 10 This is a graph showing the tumor inhibition effect of using diethylamine mesoporous silica loaded with siAURKB in a mouse subcutaneous colon cancer model, as described in Example 1.

[0037] Figure 11 This is a transmission electron microscope (TEM) image of mesoporous silica from Example 2;

[0038] Figure 12 This is a transmission electron microscope (TEM) image of mesoporous silica from Example 3;

[0039] Figure 13 This is a transmission electron microscope image of the product in Comparative Example 1. Detailed Implementation

[0040] The present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and not for limiting the scope of the invention. The raw materials and instruments used in the following embodiments are all commercially available; unless otherwise specified, the equipment and preparation processes used are conventional equipment and conventional processes. Example 1

[0041] See Figure 1 By using 25mg PS 147 -b-PAA 16 With 40mg PS 120Dissolve 100 mg of octadecyltrimethylammonium bromide in 5 mL of tetrahydrofuran to obtain mixture 1; then dissolve 100 mg of octadecyltrimethylammonium bromide in 20 mL of ultrapure water and add 250 μL of ammonia water to obtain mixture 2; then add 162 μL of tetraethyl orthosilicate to 40 mL of ethanol solution to obtain mixture 3; then stir mixture 1 evenly and pour it into mixture 2 to obtain mixture 4; then pour mixture 4 into mixture 3 to obtain mixture 5; finally, stir mixture 5 at 500 rpm for 10 min and let it stand for 18 h, then centrifuge to collect the double mesoporous silica nanoparticles, and place them in a muffle furnace to calcine at a rate of 1 °C / min to 550 °C and hold for 360 min to remove polymers and surfactants; disperse 20 mg of the collected nanoparticles in 10 mL of toluene and add 100 μL of diethylaminosilane, react at 60 °C overnight under nitrogen protection, and centrifuge to collect diethylamine mesoporous silica nanoparticles. Subsequently, diethylamine mesoporous silica nanoparticles were mixed with siRNA specifically knocking out AURKB (siAURKB) at a mass ratio of 25:1 in enzyme-free water by vortexing to prepare a dispersion with a nanoparticle concentration of 7.8 mg / mL. The dispersion was injected intratumorally into mice with subcutaneous colorectal tumors of colon cancer at a frequency of 50 μL every 3 days, ultimately achieving highly effective tumor treatment.

[0042] Example 2

[0043] The implementation method and basic formula are the same as in Example 1, except that PS is used instead of PS. 120 The dosage was reduced to 10mg.

[0044] Example 3

[0045] The implementation method and basic formula are the same as in Example 1, except that the PS is adjusted. 147 -b-PAA 16 The dosage is 50 mg, and the dosage of octadecyltrimethylammonium bromide is 50 mg.

[0046] Compare with Example 1

[0047] The implementation method and basic formula are the same as in Example 1, except that the PS is adjusted. 147 -b-PAA 16 The dosage of PS is outside the preferred range compared to the dosage of STAB. 147 -b-PAA 16 The dosage is 200mg, and the dosage of STAB is 10mg.

[0048] In Example 1, diethylamine mesoporous silica, such as Figure 2 and Figure 3 As shown in the figure, uniform silica particles with exposed labyrinthine mesopores are formed.

[0049] Figure 4 The DFT pore size distribution diagram of diethylamine mesoporous silica in Example 1 is shown. The diagram shows that the small mesopores in the diethylamine mesoporous silica are approximately 2 nm in diameter, while larger mesopores range from 10 nm to 50 nm. Furthermore, DFT analysis indicates that the pore volume of the diethylamine mesoporous silica is 1.4089 cm³. 3 / g, with a specific surface area of ​​583.2438m². 2 / g.

[0050] Figure 5 The EDS elemental analysis diagram of diethylamine mesoporous silica in Example 1 is shown. As can be seen from the figure, there is a certain amount of nitrogen on the surface of the mesoporous silica, which proves the successful modification of the diethylamine group.

[0051] Figure 6 The image shows a statistical graph illustrating the cytotoxicity of diethylamine mesoporous silica to human embryonic kidney cells (293T) in Example 1. 293T cells were seeded at a density of 5000 cells per well in 96-well plates, with 100 μL of culture medium added to each well. After incubation for 24 hours until complete cell attachment, the cells were treated with fresh culture medium containing different concentrations of diethylamine mesoporous silica and cultured for another 24 hours. Cell viability was then assessed using a CCK-8 assay kit. The CCK-8 kit was purchased from Beyotime Biotechnology. The results showed that the obtained diethylamine mesoporous silica had no significant toxicity to normal cells at high concentrations (cell viability greater than 90%).

[0052] Figure 7 The agarose gel electrophoresis image of the siRNA loading on diethylamine mesoporous silica in Example 1 shows that the diethylamine mesoporous silica nanoparticles and siRNA were vortexed in enzyme-free water at a mass ratio of 25:1, and then the loading was tested by agarose gel electrophoresis. As can be seen from the figure, there is no fluorescent band above the carrier group, indicating that the surface carrier is fully loaded with siRNA.

[0053] Figure 8 To investigate the phagocytic effect of mouse colon cancer cells (CT26 cells) on diethylamine mesoporous silica loaded with siRNA in Example 1, CT26 cells were seeded at a density of 40,000 cells per well in 12-well plates, with 1 mL of culture medium added to each well. The cells were then incubated for 24 hours until complete cell adhesion. Next, the cells were treated with serum-free medium containing diethylamine mesoporous silica loaded with Cy5-labeled siRNA (siRNA concentration: 0.8 μg / mL; diethylamine mesoporous silica concentration: 20 μg / mL) and cultured for another 6 hours. After washing with phosphate-buffered saline, the cells were collected and analyzed by flow cytometry. The results showed a significant improvement in phagocytosis compared to naked siRNA. The siRNA was purchased from Gemma Genetics.

[0054] Figure 9 This is a Western blot analysis of the AURKB knockout effect in mouse colon cancer cells using diethylamine mesoporous silica-loaded siAURKB in Example 1. CT26 cells were seeded at a density of 40,000 cells per well in 12-well plates, with 1 mL of culture medium added to each well. The cells were then incubated for 24 hours until complete cell adhesion. After discarding the old culture medium, the cells were cultured for another 48 hours in 1 mL of medium containing diethylamine mesoporous silica loaded with siAURKB (siAURKB concentration: 0.8 μg / mL, diethylamine mesoporous silica concentration: 20 μg / mL). Western blot analysis was performed using anti-AURKB antibody and actin antibody as primary antibodies, following standard procedures. The blot membrane was developed using enhanced chemiluminescence. The results showed that diethylamine mesoporous silica loaded with siAURKB had a more significant knockdown effect on AURKB compared to naked siAURKB.

[0055] Figure 10 This image shows the tumor-inhibiting effect of diethylamine mesoporous silica-loaded siAURKB on a mouse subcutaneous colon cancer model, as described in Example 1. Diethylamine mesoporous silica nanoparticles and siAURKB were vortexed in enzyme-free water at a mass ratio of 25:1 to prepare a dispersion with a nanoparticle concentration of 7.8 mg / mL. The dispersion was injected intratumorally into mice with subcutaneous colon cancer tumors at a dose of 50 μL every 3 days. The results showed that the diethylamine mesoporous silica-loaded siAURKB prepared in this invention had a significant inhibitory effect on mouse colon cancer tumors.

[0056] Figure 11 The image shows a transmission electron microscope (TEM) image of mesoporous silica from Example 2. It can be clearly seen from the image that reducing the amount of polystyrene homopolymer within the preferred range will reduce the pore size of the mesoporous silica.

[0057] Figure 12 The image shows a transmission electron microscope (TEM) image of mesoporous silica from Example 3. As can be seen from the image, changing the amount of block copolymer and surfactant within the preferred range will alter the morphology of the mesoporous silica.

[0058] Figure 13 The image shows a transmission electron microscope (TEM) image of the product in Comparative Example 1. It can be seen from the image that when the formulation is outside the preferred range, it will be impossible to successfully prepare regularly shaped mesoporous silica nanoparticles.

[0059] In summary, according to the preparation method of the present invention, RNA is loaded onto ionizable mesoporous silica, which can improve the efficiency of cellular uptake of RNA, thereby achieving better therapeutic effects.

[0060] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of the invention. Various variations can be made to the above embodiments of the present invention. All simple and equivalent changes and modifications made in accordance with the claims and description of this application fall within the protection scope of the claims of this patent. All aspects not described in detail in this invention are conventional technical content.

Claims

1. A method for preparing bimesoporous silica nanoparticles modified with ionizable amino groups, characterized in that, The preparation method includes the following steps: S1, dissolve polystyrene-polyacrylic acid block copolymer and polystyrene homopolymer in tetrahydrofuran to obtain mixture 1; S2, Dissolve the surfactant in ultrapure water and add ammonia to obtain mixture 2; S3, add the silicon source to the ethanol solution to obtain mixture 3; S4, after stirring the mixture 1 evenly, pour it into the mixture 2 to obtain the mixture 4; S5, pour mixture 4 into mixture 3 to obtain mixture 5; S6, after stirring the mixture 5, let it stand; S7, centrifugation to collect dual-mesoporous silica nanoparticles; S8. The collected double mesoporous silica nanoparticles are placed in a muffle furnace and calcined at 500-600℃ to remove polymers and surfactants. S9, the collected bimesoporous silica is dispersed in toluene and a silane coupling agent with ionizable amine groups is added to obtain mixture 6; S10, Heat and stir the mixture 6, and react under a nitrogen atmosphere; S11, centrifugation collection of bimesoporous silica nanoparticles with surface-modified ionizable amine groups.

2. The preparation method according to claim 1, characterized in that, In step S1, the polystyrene portion of the polystyrene-polyacrylic acid block copolymer has a molecular weight range of 5200 Da-41600 Da, and the polyacrylic acid portion has a molecular weight range of 720 Da-1440 Da.

3. The preparation method according to claim 1, characterized in that, In step S1, the molecular weight range of the polystyrene homopolymer is 5200 Da-41600 Da.

4. The preparation method according to claim 1, characterized in that, In step S2, the surfactant is selected from one or more of dodecyltrimethylammonium bromide, hexadecyltrimethylammonium bromide, and octadecyltrimethylammonium bromide.

5. The preparation method according to claim 1, characterized in that, The mass ratio of polystyrene-polyacrylic acid block copolymer, polystyrene homopolymer, and surfactant is (0.25-1):(0.1-1):(0.25-1).

6. The preparation method according to claim 1, characterized in that, In step S3, the silicon source is selected from one or more of tetraethyl orthosilicate, methyl orthosilicate, and sodium silicate.

7. The preparation method according to claim 1, characterized in that, In step S6, the stirring speed is 500~1300 rpm, the stirring time is 10~20 minutes, and the standing time is 10~24 hours.

8. The preparation method according to claim 1, characterized in that, In step S9, the ionizable amino group is selected from one or more of the following: diethylamine group, dimethylamine group, piperidine group, piperazine group, N-methylpiperazine group, and imidazole group.

9. A type of modified ionizable amine-modified bimesoporous silica nanoparticle prepared by the preparation method according to any one of claims 1-8, characterized in that, The nanoparticles have a dual mesoporous structure, with small mesopores having a diameter of 1-5 nm and large mesopores having a diameter of 10-50 nm.

10. A method for preparing modified ionizable amino group-modified bimesoporous silica nanoparticles according to any one of claims 1-8, or the application of modified ionizable amino group-modified bimesoporous silica nanoparticles according to claim 9 in RNA delivery.

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