Metal polyphenol network-coated drug-loaded mesoporous organic silicon nanoparticles as well as preparation method and application of metal polyphenol network-coated drug-loaded mesoporous organic silicon nanoparticles

Abstract

The invention belongs to the technical field of tumor treatment, and particularly relates to a metal polyphenol network coated drug-loaded mesoporous organic silicon nanoparticle and a preparation method and application thereof.The mesoporous organic silicon nanoparticle (MON) is prepared through a micelle / precursor co-template assembly method, then amination modification, carboxylation modification and drug co-loading are further conducted on the mesoporous organic silicon nanoparticle (MON), and the metal polyphenol network coated drug-loaded mesoporous organic silicon nanoparticle is obtained. The DQMCTH nano-particles are prepared through the steps that DQMCTH nano-particles are taken as a raw material, a copper salt solution and a tannic acid (TA) solution are added and mixed, then a coordination complexation reaction is conducted, hyaluronic acid (HA) is added for modification, and the DQMCTH nano-particles obtained through the method are in a sphere-like shape, regular in shape, uniform in distribution and free of obvious dendritic branches and central radial pore channels. The polypeptide is effectively protected by MPN and is subjected to HA targeted modification; more importantly, the DQMCTH nanoparticles prepared by the method disclosed by the invention show pH / GSH / HAase triple sensitive drug release, and cell and animal experiment results show that the DQMCTH nanoparticles prepared by the method disclosed by the invention have relatively high anti-tumor activity on a drug-resistant breast cancer model.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of tumor treatment, and particularly relates to metal polyphenol network coated drug-loaded mesoporous organosilica nanoparticles as well as a preparation method and application thereof. BACKGROUND

[0002] Tumor multidrug resistance (MDR) is the main cause of chemotherapy failure, which is driven by a dynamic network composed of multiple cellular components and abnormal signaling pathways in the tumor microenvironment, leading to treatment failure through enhanced drug efflux, activated DNA repair and apoptosis escape. Traditional therapy is difficult to overcome MDR, while the nano precise delivery system breaks through the bottleneck by virtue of the synergistic effect of controllable drug release, inhibition of efflux pump and remodeling of immune metabolism, showing a significant clinical transformation prospect.

[0003] Natural flavonoid compound quercetin (Que) has become a research hotspot due to its unique multi-target reverse MDR effect. Its mechanism includes significantly improving intracellular accumulation of chemotherapeutic drugs, down-regulating expression of drug resistance related proteins, inhibiting tumor invasion and metastasis, activating mitochondrial apoptosis pathway and inhibiting heat shock protein mediated survival signals, etc. It is worth noting that Que has a bidirectional protective effect, that is, while enhancing the killing of tumor cells, it can also reduce the toxic side effects of chemotherapeutic drugs on normal tissues such as heart (e.g., reducing the incidence of doxorubicin cardiomyopathy). However, the inherent defects of Que, such as poor water solubility, low oral bioavailability, and easy oxidation and degradation, seriously limit its clinical application value. Mesoporous organosilica nanoparticles (MONs) have become an ideal platform for co-loading Que and doxorubicin (DOX) due to their tunable pore size, high specific surface area and easily modified surface. Among them, redox-responsive MONs bridged by four-sulfur bonds can be specifically disintegrated in the high glutathione (GSH) microenvironment of tumors, realizing a triple synergistic effect: (1) drug burst release caused by carrier disintegration; (2) intracellular GSH consumption (>80%) caused by four-sulfur bond cleavage; (3) disruption of redox homeostasis caused by GSH depletion, which synergistically enhances reactive oxygen species (ROS)-driven ferroptosis and chemodynamic therapy (CDT). However, the mesoporous structure of MONs is prone to premature drug leakage in the body circulation. Current gating strategies such as polymer plugging have problems of response lag and complex process. Recent studies have shown that metal-polyphenol networks (MPN) can provide an ideal cap for MON channels due to their pH responsiveness, super strong adhesion and adjustable photothermal / CDT activity. This system self-assembles a dense protective layer through metal ion and polyphenol coordination, which can effectively block drug leakage and be specifically activated to release drugs in the tumor microenvironment, laying the core design foundation for a new generation of intelligent drug delivery systems. SUMMARY

[0004] The purpose of the present application is to provide a metal-polyphenol network coated drug-loaded mesoporous organosilica nanoparticle and its preparation method and application.

[0005] To achieve the above purpose, the technical scheme adopted by the present application is: The preparation method of the metal-polyphenol network coated drug-loaded mesoporous organosilica nanoparticle comprises the following steps: (1) Preparation of mesoporous organosilica nanoparticles: In a reaction bottle, add a CTAC and TEA aqueous solution, and stir magnetically at 80°C. Define as solution A. Take TEOS and BTES, mix and ultrasonic, define as solution B. Slowly add solution B to solution A, and stir magnetically at 80°C. After stirring, centrifuge, discard the supernatant, and wash with anhydrous ethanol. Disperse the reaction product in NaCl-methanol, stir, centrifuge, wash with anhydrous ethanol, and freeze-dry to collect the precipitate to obtain MON.

[0006] (2) Preparation of drug-loaded mesoporous organosilica nanoparticles: The above obtained MON was dispersed in water, APTES was added, and magnetic stirring was performed. After centrifugation, the supernatant was discarded, and the precipitate was collected by washing with ultrapure water to obtain amino-modified MON (MON-NH2). Succinic acid (SA) was added to EDC and NHS in ethanol for activation. The SA solution was added to the above prepared MON-NH2, and magnetic stirring was performed. After centrifugation, the precipitate was collected by washing with purified water and ethanol in sequence, and the precipitate was collected to obtain carboxyl-modified MON (MON-COOH). The DOX / Que ethanol solution was added to the above prepared MON-COOH, and stirring was performed in the dark for 24 h. After centrifugation, the precipitate was collected by washing with ethanol to obtain the MON nanoparticles co-loaded with DOX and Que (DQM).

[0007] (3) Preparation of metal polyphenol network coated drug-loaded mesoporous organosilica nanoparticles: The DQM nanoparticles prepared in step (2) were ultrasonically dispersed in purified water, and CuCl2 solution was added. After stirring in the dark, tannic acid (TA) solution was added, and stirring was performed in the dark. After centrifugation, the precipitate was washed with purified water to obtain DQM nanoparticles coated with MPN formed by Cu and TA (DQMCT nanoparticles). The DQMCT nanoparticles were dispersed in hyaluronic acid (HA) solution, and stirring was performed in the dark. After centrifugation, the precipitate was washed with purified water to obtain HA-modified DQMCT nanoparticles (DQMCTH nanoparticles).

[0008] Further, in step (2), the volume ratio of SA to MON-NH2 is 1:1-20.

[0009] Further, in step (2), the mass ratio of DOX / Que ethanol solution to MON-COOH is 0.5-1:1-20.

[0010] Further, in step (2), the total concentration of DOX / Que ethanol solution ranges from 200 to 600 μg / mL.

[0011] Further, in step (3), the mass ratio of TA to Cu is 1:1, and the concentration of TA ranges from 1.2 to 60.0 mM.

[0012] Further, in step (3), the concentration of HA ranges from 0.1 to 2.0 mg / mL.

[0013] Another object of the present application is to provide a DQMCTH nanoparticle, i.e., a hyaluronic acid-modified DQMCT nanoparticle prepared by the above method.

[0014] Another object of the present application is to provide a hyaluronic acid-modified DQMCT nanoparticle for use in the preparation of a drug for treating drug-resistant breast cancer.

[0015] The advantages of this invention are: 1. The method for preparing DQMCTH nanoparticles provided by the present invention firstly prepares mesoporous organosilicon nanoparticles (MON) by micelle / precursor co-mold assembly method, then further modifies them by amylation, carboxylation, and co-loading with drugs, and finally adds copper salt solution and tannic acid (TA) solution to carry out coordination complexation reaction, and finally adds hyaluronic acid (HA) for modification to obtain the final product. 2. The DQMCTH nanoparticles obtained by the method of this invention are spherical, with regular morphology and uniform distribution, without obvious dendritic branches and central radial channels. The method of this invention can effectively load drugs and is effectively protected by MPN and targeted by HA. More importantly, the DQMCTH nanoparticles prepared by the method of this invention exhibit triple-sensitive drug release of pH / GSH / HAase (pH sensitivity is due to pH-responsive degradation of MPN (middle layer), GSH sensitivity is due to GSH-responsive disintegration of MPN (middle layer) and MON (core), and HAase sensitivity is due to HAase-responsive degradation of HA (outer layer). This may be due to the synergistic effect of HA-mediated HAase response, MPN-mediated pH / GSH dual-responsive disintegration, and MON GSH-responsive disintegration. In addition, the DQMCTH nanoparticles prepared by the method of this invention also have high stability and biocompatibility, which may be related to the MPN and HA modification of the outermost layer of the nanoparticles. 3. Cell and animal experiments showed that the DQMCTH nanoparticles prepared by the method of this invention have high anti-tumor activity against drug-resistant breast cancer models. This may be related to its mediated disruption of the redox homeostasis of drug-resistant tumor cells. Specifically, the DQMCTH nanoparticles disrupt redox homeostasis through a dual action of "GSH depletion-Fenton-like reaction amplification of ROS", thereby enhancing the therapeutic sensitivity of drug-resistant cells. Attached Figure Description

[0016] Figure 1 Characterization of the MON-COOH nanoparticles prepared in Examples 1-5: (A) Hydrated particle size; (B) Polydispersity index; (C) Potential.

[0017] Figure 2 The encapsulation efficiency and drug loading of the DQM nanoparticles prepared in Examples 6-11 are shown.

[0018] Figure 3 The encapsulation efficiency and drug loading of the DQM nanoparticles prepared in Examples 12-16 are shown.

[0019] Figure 4 Characterization of the DQMCT nanoparticles prepared in Examples 17-24: (A) Hydrated particle size; (B) Polydispersity index; (C) Potential.

[0020] Figure 5 Characterization of the DQMCTH nanoparticles prepared in Examples 25-30: (A) Hydrated particle size; (B) Polydispersity index; (C) Potential.

[0021] Figure 6 The cumulative drug release curve of the DQMCTH nanoparticles in Example 31 is shown.

[0022] Figure 7 The results of stability tests of DQMCTH nanoparticles in different media in Example 32 are shown; (A) Ultrapure water; (B) Complete culture medium.

[0023] Figure 8 The results are the hemolytic activity assay results of the DQMCTH nanoparticles in Example 33.

[0024] Figure 9 The inhibition rate of DQMCTH nanoparticles on MCF-7 cells and MCF-7 / DOX cells in Example 34 is shown.

[0025] Figure 10 The percentage of GSH consumed by MCF-7 / DOX cells in the DQMCTH nanoparticles in Example 35 is the percentage of GSH consumed by the MCF-7 / DOX cells.

[0026] Figure 11 The ROS generated by the DQMCTH nanoparticles in Example 35 on MCF-7 / DOX cells.

[0027] Figure 12 The curve shows the relative tumor volume change of the drug-resistant breast cancer subcutaneous tumor model in Example 36 after treatment with DQMCTH nanoparticles.

[0028] Figure 13 This is a pathological section analysis of the drug-resistant breast cancer subcutaneous tumor model in Example 36 after treatment with DQMCTH nanoparticles.

[0029] Figure 14 The image shows the GSH consumption of the drug-resistant breast cancer subcutaneous tumor model in Example 36 after treatment with DQMCTH nanoparticles.

[0030] Figure 15 The ROS production in the drug-resistant breast cancer subcutaneous tumor model in Example 36 after treatment with DQMCTH nanoparticles is shown. Detailed Implementation

[0031] Example 1 Preparation of carboxyl-modified mesoporous organosilicon nanoparticles (MON-COOH nanoparticles): Weigh 0.5 g of hexadecyltrimethylammonium chloride and 0.06 g of triethanolamine into a 50 mL beaker. Add 10 mL of purified water and stir until homogeneous at room temperature, then dilute to 20 mL (defined as solution A). Accurately measure 1 mL of TEOS and 0.2 mL of BTES (defined as solution B), mix, and sonicate for 30 min. Preheat the magnetic stirrer to 80℃ and set the speed to 500 r / min. Transfer solution A to the 80℃ magnetic stirrer and slowly add solution B dropwise to solution A over 20 min. After the reaction is complete, continue stirring on the magnetic stirrer for 4 h. After stirring, centrifuge (15000 rpm × 20 min), discard the supernatant, add anhydrous ethanol to the precipitate, sonicate to disperse, and centrifuge again (10000 rpm × 10 min). Repeat this operation twice. The MON obtained from the reaction was dispersed in 20 mL of template removal agent (NaCl-methanol (8 mg / mL), stirred for 12 h, and centrifuged (10000 rpm × 10 min). This operation was repeated several times. The supernatant was discarded, and the precipitate was dispersed in NaCl-methanol solution and centrifuged (10000 rpm × 10 min). This operation was repeated twice. After the last time, the precipitate was washed twice with ethanol, centrifuged, the supernatant was discarded, and the precipitate was collected by freeze-drying to obtain MON.

[0032] Accurately weigh 5 mg of dried MON and dissolve it in 10 mL of purified water. Add 0.1 mL of APTES and place the mixture on a magnetic stirrer. Stir at 300 r / min for 8 h until the reaction is complete. Centrifuge (10000 rpm × 10 min), discard the supernatant, wash the precipitate with purified water, disperse, and centrifuge (10000 rpm × 10 min). Repeat the above operation twice. After the last washing, wash twice with ethanol, centrifuge, discard the supernatant, collect the precipitate, and obtain the amino-modified MON, i.e., MON-NH2. Disperse the precipitate in 5 mL of anhydrous ethanol for later use. Add succinic acid (SA) to ethanol containing 39 mg EDC and 39 mg NHS and activate under ice bath conditions for 1 h. The MON-NH2 prepared above was added to SA solution (the volume ratio of succinic acid solution to MON-NH2 was 1:1), and the reaction was carried out at room temperature for 24 h. After centrifugation (10000 rpm × 10 min), the precipitate was collected and washed twice with purified water and twice with ethanol. The precipitate was collected to obtain carboxylated modified MON, i.e., MON-COOH.

[0033] Example 2 Replace the volume ratio of SA solution and MON-NH2 nanoparticle dispersion in step (2) of Example 1 with 1:2, and keep all other parameters the same as in Example 1.

[0034] Example 3 The volume ratio of SA solution and MON-NH2 nanoparticle dispersion in step (2) of Example 1 was replaced with 1:4, and all other parameters were the same as in Example 1.

[0035] Example 4 The volume ratio of SA solution and MON-NH2 nanoparticle dispersion in step (2) of Example 1 was replaced with 1:6, and all other parameters were the same as in Example 1.

[0036] Example 5 The volume ratio of SA solution and MON-NH2 nanoparticle dispersion in step (2) of Example 1 was replaced with 1:8, and all other parameters were the same as in Example 1.

[0037] The hydrated particle size, polydispersity index, and potential of MON-COOH prepared in Examples 1-5 were measured, and the results are as follows: Figure 1 As shown, when the MON-NH2 ratio is low (e.g., 1:1), SA may be excessive, leading to excessive intermolecular cross-linking and a significant increase in particle size. Conversely, when the MON-NH2 ratio is too high (e.g., ≥1:6), the SA coating is relatively insufficient, making it difficult to achieve complete coating of single particles. Instead, it promotes the "bridging" effect of multiple MON-NH2 nanoparticles through the same SA molecule, causing severe aggregation, manifested as a sharp increase in particle size and a significant increase in polydispersity index (PDI). Under the condition of a ratio of 1:2, SA can achieve sufficient and uniform surface coating of MON-NH2 and complete effective carboxylation modification, forming a complete and appropriately thick shell structure, thereby obtaining MON-COOH nanoparticles with the smallest particle size and the most concentrated distribution (lowest PDI). This indicates that at this ratio, a better balance is achieved between SA and MON-NH2. Therefore, the present invention preferably uses a volume ratio of SA solution to MON-NH2 nanoparticle dispersion of 1:2, and the MON-COOH nanoparticles prepared under this condition are used for subsequent research.

[0038] Example 6 The DOX / Que ethanol solution was added to the MON-COOH prepared in Example 2, with a drug-to-MON-COOH mass ratio of 0.5:1. The mixture was stirred in the dark for 24 h, centrifuged, washed with ethanol, and the precipitate was collected to obtain MON nanoparticles co-loaded with DOX and Que (DQM nanoparticles).

[0039] Example 7 The mass ratio of drug to MON-COOH in Example 6 was replaced with 1:1, and all other parameters were the same as in Example 6.

[0040] Example 8 The mass ratio of the drug to MON-COOH in Example 6 was replaced with 1:2, and all other parameters were the same as in Example 6.

[0041] Example 9 The mass ratio of the drug to MON-COOH in Example 6 was replaced with 1:5, and all other parameters were the same as in Example 6.

[0042] Example 10 The mass ratio of drug to MON-COOH in Example 6 was replaced with 1:10, and all other parameters were the same as in Example 6.

[0043] Example 11 The mass ratio of drug to MON-COOH in Example 6 was replaced with 1:20, and all other parameters were the same as in Example 6.

[0044] The encapsulation efficiency and drug loading of the DQM nanoparticles prepared in Examples 6-11 were measured, and the results are as follows: Figure 2 As shown. The specific test steps for encapsulation efficiency and drug loading are as follows: The DQM nanoparticles prepared in Examples 6-11 were centrifuged at 14000 rpm for 30 min at 4℃. The supernatant was collected and washed again with ethanol. The supernatant was collected again by centrifugation under the same conditions. The supernatants collected twice were combined, and the absorbance of free Que and the fluorescence intensity of free DOX were measured by UV spectrophotometer and fluorescence spectrophotometer, respectively. The content of free drug was calculated and recorded as Wfree, the initial amount added was recorded as W1, and the mass of DQM after drying was recorded as W. The encapsulation efficiency was calculated according to the formula: Encapsulation efficiency (%) = [(W1 - Wfree) / W1] × 100%. The drug loading was calculated according to the formula: Drug loading (%) = [(W1 - Wfree) / W] × 100%. Each example was measured 3 times, and the average value was taken. From Figure 2 As can be seen, with the increase of the carrier ratio, the encapsulation efficiency remained above 85% and showed a gradual upward trend, while the drug loading showed a pattern of first increasing and then decreasing. The encapsulation efficiency reached its maximum when the drug-to-carrier mass ratio was 1:10; the drug loading was highest when the drug-to-carrier mass ratio was 1:1. Considering the balance between encapsulation efficiency and drug loading, this invention preferably uses a drug-to-MON-COOH mass ratio of 1:1. DQM nanoparticles prepared under this condition were used for subsequent research.

[0045] Example 12 The DOX / Que ethanol solution was added to the MON-COOH prepared in Example 2, with a drug-to-MON-COOH mass ratio of 1:1 and a total concentration of 200 μg / mL for the DOX / Que mixed solution. The mixture was stirred in the dark for 24 h, centrifuged, washed with ethanol, and the precipitate was collected to obtain MON nanoparticles co-loaded with DOX and Que (DQM nanoparticles).

[0046] Example 13 The total concentration of the DOX / Que mixed solution in Example 12 was replaced with 300 μg / mL, and all other parameters were the same as in Example 12.

[0047] Example 14 The total concentration of the DOX / Que mixed solution in Example 12 was replaced with 400 μg / mL, and all other parameters were the same as in Example 12.

[0048] Example 15 The total concentration of the DOX / Que mixed solution in Example 12 was replaced with 500 μg / mL, and all other parameters were the same as in Example 12.

[0049] Example 16 The total concentration of the DOX / Que mixed solution in Example 12 was replaced with 600 μg / mL, and all other parameters were the same as in Example 12.

[0050] Using the same test method, the encapsulation efficiency and drug loading of the preparations in Examples 12-16 were measured, and the results are as follows: Figure 3 As shown, the encapsulation efficiency of DQM nanoparticles prepared at different drug concentrations remained at a high level of 93% to 96%, with minimal fluctuations, indicating that the encapsulation efficiency was not significantly affected by changes in drug concentration. However, when the total concentration of the two drugs was 300 μg / mL, the prepared DQM nanoparticles exhibited the highest drug loading. Based on the optimization results of this key parameter of drug loading, this method preferentially uses a total concentration of 300 μg / mL for the DOX / Que mixed solution, and the DQM nanoparticles prepared under this condition were used for subsequent studies.

[0051] Example 17 The DQM nanoparticles prepared in Example 13 were ultrasonically dispersed and dissolved in purified water. A CuCl2 solution with a concentration of 1.2 mM was added, and the mixture was stirred at room temperature in the dark for 12 h. Then, an equal volume of TA solution with the same concentration was added, and the mixture was stirred at room temperature in the dark for 30 min. The mixture was then centrifuged at 10,000 rpm for 10 min, and the precipitate was washed twice with purified water to obtain MPN-coated DQM nanoparticles (DQMCT nanoparticles) formed by Cu and TA.

[0052] Example 18 The concentration of CuCl2 solution in Example 17 was replaced with 2.4 mM, and all other parameters were the same as in Example 17.

[0053] Example 19 The concentration of the CuCl2 solution in Example 17 was replaced with 4.8 mM, and all other parameters were the same as in Example 17.

[0054] Example 20 The concentration of the CuCl2 solution in Example 17 was replaced with 9.6 mM, and all other parameters were the same as in Example 17.

[0055] Example 21 The concentration of the CuCl2 solution in Example 17 was replaced with 24.0 mM, and all other parameters were the same as in Example 17.

[0056] Example 22 The concentration of the CuCl2 solution in Example 17 was replaced with 36.0 mM, and all other parameters were the same as in Example 17.

[0057] Example 23 The concentration of the CuCl2 solution in Example 17 was replaced with 48.0 mM, and all other parameters were the same as in Example 17.

[0058] Example 24 The concentration of the CuCl2 solution in Example 17 was replaced with 60.0 mM, and all other parameters were the same as in Example 17.

[0059] The hydration size, polydispersity index, and potential of the DQMCT nanoparticles prepared in Examples 17-24 were measured, and the results are as follows: Figure 4 As shown, within the CuCl2 solution concentration range of 2.4 mM to 48 mM, the hydrodynamic diameter of DQMCT nanoparticles remained relatively small, and the polydispersity index (PDI) was generally below 0.2, indicating good monodispersity within this concentration range. When the CuCl2 solution concentration was further increased to 60 mM, the particle size significantly increased to 212.3 nm, suggesting that high concentrations may lead to some degree of nanoparticle aggregation or Ostwald ripening. In the low CuCl2 solution concentration range (<4.8 mM), the absolute value of the Zeta potential was approximately 16 mV, indicating that the DQMCT nanoparticle dispersion system was in a relatively stable state. When the concentration was 4.8 mM, the Zeta potential was close to zero (–0.09 mV), which is the isoelectric point of the system. Electrostatic repulsion was weakest at this point, and nanoparticles were prone to aggregation or flocculation, resulting in poor stability. When the CuCl2 solution concentration was higher than 4.8 mM, the absolute value of the Zeta potential was greater than 20 mV, indicating that the electrostatic repulsion between particles was enhanced, and the stability of the dispersion system was significantly improved. In summary, DQMCT nanoparticles prepared with a CuCl2 solution concentration of 48 mM simultaneously possess a high absolute Zeta potential (good electrostatic stability), a low PDI (uniform particle size distribution), and a suitable hydrodynamic diameter. Therefore, this invention preferably uses 48 mM as the CuCl2 solution concentration, and DQMCT nanoparticles prepared under this condition are used for subsequent experiments.

[0060] Example 25 The DQMCT nanoparticles prepared in Example 23 were redispersed in 5 mL of HA solution (HA concentration was 0.1 mg / mL), stirred at room temperature in the dark for 6 h, centrifuged at 10000 rpm for 10 min, and the precipitate was washed twice with purified water to obtain HA-modified DQMCT nanoparticles (DQMCTH nanoparticles).

[0061] Example 26 The concentration of the HA solution in Example 24 was replaced with 0.25 mg / mL, and all other parameters were the same as in Example 24.

[0062] Example 27 The concentration of the HA solution in Example 24 was replaced with 0.50 mg / mL, and all other parameters were the same as in Example 24.

[0063] Example 28 The concentration of the HA solution in Example 24 was replaced with 0.75 mg / mL, and all other parameters were the same as in Example 24.

[0064] Example 29 The concentration of the HA solution in Example 24 was replaced with 1.0 mg / mL, and all other parameters were the same as in Example 24.

[0065] Example 30 The concentration of the HA solution in Example 24 was replaced with 2.0 mg / mL, and all other parameters were the same as in Example 24.

[0066] The hydration size, polydispersity index, and potential of the DQMCTH nanoparticles prepared in Examples 25-30 were measured, and the results are as follows: Figure 5As shown, when the concentration of HA solution ranges from 0.1 to 0.75 mg / mL, the hydrodynamic diameter of the prepared DQMCTH nanoparticles gradually decreases, reaching a minimum at 0.75 mg / mL. Simultaneously, the polydispersity index (PDI) also decreases to its lowest value, indicating that the DQMCTH nanoparticles prepared at this concentration have optimal particle size and monodispersity. However, when the HA solution concentration increases to 1.0 mg / mL, the particle size significantly increases to 345.8 nm, suggesting significant aggregation or agglomeration of the nanoparticles under high concentration conditions. At a HA solution concentration of 0.25 mg / mL, the Zeta potential is -38.8 mV, indicating excellent electrostatic stability. With increasing HA solution concentration, the absolute value of the Zeta potential decreases, but it remains at -25.9 mV at 0.75 mg / mL, within a good stability range. When the HA solution concentration was further increased to 2.0 mg / mL, the Zeta potential dropped to -7.8 mV, close to the isoelectric point of the system. The electrostatic repulsion significantly weakened, making the nanoparticles prone to aggregation and flocculation, leading to a sharp decrease in the stability of the dispersion system. In summary, the nanoparticles prepared with an HA solution concentration of 0.75 mg / mL possess the smallest hydrodynamic diameter, optimal monodispersity (low PDI), and good Zeta potential stability, while effectively avoiding the aggregation risk in high-concentration regions and the instability near the isoelectric point. Therefore, this invention preferably uses an HA solution concentration of 0.75 mg / mL, and the DQMCTH nanoparticles prepared under these conditions are used for subsequent research.

[0067] Example 31 Determination of the release behavior of DQMCTH nanoparticles DQMCTH nanoparticles prepared in Example 28 were placed in different dialysis bags (MWCO = 3500 Da), tied at both ends, and then placed into centrifuge tubes containing 20 mL of buffer solutions with different pH values ​​(pH 7.4, 6.5, 5.0), containing GSH (0, 10 mM), or containing HAase (0, 110 IU / mL). The tubes were incubated in the dark at 37°C and 100 rpm in a constant-temperature shaker. Samples were taken at 0.5, 1, 2, 4, 6, 8, 10, 12, 24, and 48 h, and an equal volume of fresh release medium at the same temperature was added simultaneously. Three replicates were prepared for each group. The concentrations of Que and DOX in each medium were determined by UV spectrophotometry and fluorescence spectrophotometry, and the cumulative release percentage was calculated.

[0068] The results are as follows Figure 6As shown, at pH = 7.4, the cumulative release rate of DQMCTH nanoparticles after 48 h was only 23.58%, while at pH = 6.5 and 5.0, the cumulative release rates reached 43.41% and 58.12%, respectively, indicating that the nanoparticles prepared in this study have good pH responsiveness. This may be due to the fact that the Cu-TA metal polyphenol network in the nanoparticles and the coordination between Cu and the drugs (DOX and Que) all have pH-responsive degradation characteristics. Compared with the cumulative release rate of DQMCTH nanoparticles in different pH release media without GSH, the cumulative release rate of DQMCTH nanoparticles in release media containing 10 mM GSH was significantly increased, indicating that the presence of GSH can effectively promote drug release. This may be due to the fact that the Cu-TA metal polyphenol network in the nanoparticles, the coordination between Cu and the drugs (DOX and Que), and the carrier MON all have GSH-responsive degradation characteristics. Similarly, compared with the cumulative drug release rate in different pH release media without HAase, the cumulative drug release rate increased to some extent after the presence of HAase. This is mainly attributed to the fact that the outermost HA-modified layer of the nanoparticles can be specifically enzymatically hydrolyzed by HAase. In summary, the DQMCTH nanoparticles prepared in this invention exhibit good pH / GSH / HAase triple sensitivity.

[0069] Example 32 Stability testing of DQMCTH nanoparticles Using particle size and drug encapsulation efficiency as indicators, the preliminary stability of DQMCTH nanoparticles in water and complete culture medium containing 10% serum was investigated for 7 consecutive days at 4°C.

[0070] The results are as follows Figure 7 As shown, the particle size and encapsulation efficiency of DQMCTH nanoparticles did not change significantly in water and complete culture medium containing 10% serum, indicating that the nanoparticles have good stability.

[0071] Example 33 Hemolytic activity test of DQMCTH nanoparticles Add 2% red blood cell suspension, PBS buffer, Triton X-100, and DQMCTH nanoparticles of different concentrations to a test tube in sequence, shake well, and immediately incubate in a constant temperature water bath at 37 (±0.5)℃ for 4 h. Centrifuge at 1500 rpm for 10 min, take the supernatant, and record the absorbance of each group at 540 nm. PBS and Triton X-100 are used as negative and positive controls, respectively. Calculate the hemolysis rate (%) according to the formula: Hemolysis rate (%) = (A-APBS) / (ATriton X100-APBS)×100%, where A is the absorbance value of the experimental group (i.e., DQMCTH nanoparticles), APBS is the absorbance value of the negative control group (i.e., PBS), and ATriton X-100 is the absorbance value of the positive control group (i.e., Triton X-100).

[0072] The results are as follows Figure 8 As shown, after DQMCTH nanoparticles of different concentrations were placed in a constant temperature incubator at 37℃ for 4 h, all red blood cell suspensions sank and the stratification interface was obvious. Moreover, the hemolysis rate of all concentrations was less than 3%, indicating that the hemolytic effect of DQMCTH nanoparticles is negligible, that is, it has good biocompatibility in mice and meets the requirements for injection.

[0073] Example 34 Evaluation of the antitumor effect of DQMCTH nanoparticles in drug-resistant breast cancer cells Logarithmic growth phase MCF-7 and MCF-7 / DOX cells were centrifuged in centrifuge tubes, the original culture medium was discarded, and an appropriate amount of fresh culture medium was added, mixed well, and seeded into 96-well plates at 100 μL per well, with a cell density of 5 × 10⁶ cells per well. 3 Add 200 μL of sterile PBS buffer to each well and place in an incubator. After overnight culture until cell attachment, aspirate the original culture medium from the wells. Treat cells with MON, MCTH, DQM, and DQMCTH, respectively, and incubate for 48 h. Afterward, aspirate the original culture medium from the wells, add 100 μL of 10% CCK-8 solution to each well, shake thoroughly, and continue culturing in the 96-well plate for 2 h. Measure the absorbance at 450 nm using a microplate reader to determine the cell inhibition rate.

[0074] The results are as follows Figure 9As shown, different concentrations of nanoparticles exhibited varying inhibitory rates on MCF-7 and MCF-7 / DOX cells. The blank carrier MON showed no significant cytotoxicity (<5%) to either cell type, while MPN-modified MCTH nanoparticles showed low cytotoxicity to both. Drug-loaded DQM and DQMCTH nanoparticles were significantly cytotoxic to both cell types, with significantly higher cytotoxicity to MCF-7 cells than to MCF-7 / DOX cells. However, in MCF-7 cells, there was no significant difference in cytotoxicity between DQM and DQMCTH nanoparticles, while a significant difference was observed in MCF-7 / DOX cells. This is because, compared to MCF-7 cells, DQM and DQMCTH nanoparticles showed a greater difference in amplifying oxidative stress levels in MCF-7 / DOX cells (experimental data included GSH consumption and ROS induction).

[0075] Example 35 Investigating the antitumor mechanism of DQMCTH nanoparticles in drug-resistant breast cancer cells.

[0076] GSH Consumption: GSH levels in MCF-7 and MCF-7 / DOX cells were measured using a GSH / GSSG assay kit. Experimental groupings were the same as in Example 34. First, a series of solutions were prepared according to the kit instructions: 10 mM GSSG stock solution was serially diluted to 0.5–15 μM standard using protein removal reagent M solution, and GSH removal helper and working solution were added. Cells were incubated at 25°C for 1 h, and absorbance was measured at 412 nm to plot a GSSG standard curve. Cells were collected 24 h after drug intervention. The supernatant was discarded, and the cells were washed with PBS, digested with trypsin, and centrifuged to obtain a cell pellet. Protein removal reagent M solution was added at three times the pellet volume, and cells were lysed using two freeze-thaw cycles in liquid nitrogen and a 37°C water bath. Cells were centrifuged at 12,000 rpm for 10 min at 4°C, and the supernatant was collected. A portion of the supernatant was used directly to determine total glutathione, while the other portion was added to the GSH removal reagent for specific determination of GSSG levels. Finally, samples were added to 96-well plates, and the total glutathione detection working solution was added and reacted for 5 min. Then, NADPH solution was added to start the reaction, and the absorbance was immediately measured at 412 nm using a microplate reader. The concentrations of total glutathione and GSSG in the samples were calculated based on the standard curve, thereby estimating the content of reduced GSH with antioxidant activity.

[0077] ROS content determination: Intracellular ROS content in MCF-7 cells and MCF-7 / DOX cells was determined using a ROS detection kit, with experimental groups as described above. First, following the manufacturer's instructions, DCFH-DA solution was diluted 1:1000 with serum-free culture medium to prepare a 10 μM DCFH-DA solution. After incubation for 24 h, the culture medium was removed, and 250 μL of the prepared DCFH-DA solution was added to each well. The cells were incubated at 37°C for 20 min, followed by washing the cells three times with serum-free cell culture medium to thoroughly remove any DCFH-DA that had not yet entered the cells. Subsequently, fluorescence signals were observed under a fluorescence microscope, and images were acquired and analyzed.

[0078] The percentage of GSH consumed by different agents in MCF-7 cells and MCF-7 / DOX cells after 24 h are as follows: Figure 10 As shown in the figure, compared with the control group, the percentage of GSH digestion was significantly increased in all nanoparticles except for the blank vector MON, indicating that DQMCTH nanoparticles can effectively consume GSH in tumor cells. It is noteworthy that the difference between DQM nanoparticles and DQMCTH nanoparticles was small in MCF-7 cells, while the difference was larger in MCF-7 / DOX cells.

[0079] Twenty-four hours after drug administration, intracellular ROS in each group of cells were detected using the reactive oxygen species fluorescent probe DCFH-DA. Fluorescence images are shown below. Figure 11 The results showed that the fluorescence intensity of each group of MCF-7 cells and MCF-7 / DOX cells was: Control group < MON group < MCTH group < DOX group < DOX + Que group < DQM group < DQMCTH group. The large amount of green fluorescence produced by the DQMCTH nanoparticle group indicated that the nanoparticles could produce a large amount of ROS after acting on tumor cells, which is considered to be due to the disintegration of MPN, MON and Cu 2+ The synergistic effect with DOX induced the generation of a large amount of ROS.

[0080] Example 36 Evaluation of the antitumor effect of DQMCTH nanoparticles in a drug-resistant breast cancer subcutaneous tumor model.

[0081] The concentration of well-growing, drug-resistant MCF-7 / DOX cells was adjusted to 3 × 10⁻⁶. 7 Cells per mL were injected subcutaneously into the right axilla of nude mice at a concentration of 0.1 mL. The mice were then housed in an SPF-grade environment with free access to food and water, and tumor growth was monitored regularly. Tumors were allowed to grow to 80–100 mm in size. 3 When the value is around 100, it indicates that the MCF-7 / DOX tumor-bearing nude mouse model has been successfully constructed.

[0082] To investigate the in vivo antitumor effect of DQMCTH nanoparticles, successfully modeled MCF-7 / DOX-bearing nude mice were randomly divided into 6 groups: ①NS group, ②free DOX group, ③free DOX + Que group, ④DQM group, ⑤MCTH group, and ⑥DQMCTH group. Each group of tumor-bearing mice was then injected intravenously with the corresponding solution every 2 days for a total of 5 administrations. The DOX concentration was 2 mg / kg. Before each administration, the tumor volume of each group of tumor-bearing mice was measured with calipers, and tumor growth curves were plotted. After treatment, the tumor-bearing mice were sacrificed, and tumor tissue was collected, embedded in paraffin, and subjected to HE staining and TUNEL assay to detect tumor cell apoptosis, thus assessing the antitumor effect of DQMCTH nanoparticles.

[0083] The results are as follows Figure 12 As shown, compared with the NS group and the free drug group, the tumor volume of tumor-bearing mice in the DQMCTH nanoparticle group was significantly reduced with increasing dosing frequency, indicating that DQMCTH nanoparticles have a significant inhibitory effect on tumor growth. HE staining results ( Figure 13 The results showed that the tumor cells in the NS and DOX groups had higher density and more intact morphology, indicating that the tumor cells were growing at a normal level. The tumor cells in the DQMCTH nanoparticle group showed the most severe damage. Furthermore, TUNEL staining of tumor tissue sections from different treatment groups further verified the apoptosis of tumor cells. The tumor cells in the NS group were uniformly arranged with almost no apoptotic cells. The DQMCTH nanoparticle group had the highest number of brown apoptotic cells, indicating that the DQMCTH nanoparticles had the strongest anti-tumor effect. This is because the DQMCTH nanoparticles not only have tumor targeting (HA-mediated), but also respond to pH / GSH / Haase in the tumor microenvironment to precisely determine whether it is a drug. They can also enhance efficacy through the synergistic effects of MON consuming GSH, chemotherapy (DOX and Que), and chemokinetic therapy (MPN undergoing a Fenton-like reaction to generate ROS).

[0084] like Figure 14 As shown, the percentage of GSH consumption in tumor tissues of mice in each experimental group increased in a gradient after treatment. Compared with the NS group, the GSH consumption percentage in the DQMCTH nanoparticle group was as high as 81.27%, indicating that it could significantly deplete GSH in tumor tissues. ROS staining results ( Figure 15 The results showed that the NS group exhibited almost no red fluorescence; the free DOX group showed weak fluorescence, while the combination of DOX and Que enhanced the fluorescence intensity, suggesting that Que can partially reverse drug resistance. The DQMCTH nanoparticle group showed the strongest red fluorescence, indicating the highest level of ROS generation. This is mainly attributed to its multiple synergistic mechanisms, including pH / GSH / HAase triple-responsive drug release, Que-mediated chemosensitization, and CDT.

Claims

1. A method for preparing drug-loaded mesoporous organosilicon nanoparticles coated with a metal polyphenol network, characterized in that, Includes the following steps: S1. Preparation of mesoporous organosilicon nanoparticles: Hexadecyltrimethylammonium chloride and triethanolamine aqueous solution were added to the reaction vessel and stirred to define solution A; tetraethyl orthosilicate and bis-(γ-triethoxysilylpropyl)tetrasulfide were mixed to define solution B; solution B was added dropwise to solution A, stirred, centrifuged, the supernatant was discarded, and the product was washed with anhydrous ethanol. The reaction product was dispersed in NaCl-methanol, stirred, centrifuged, washed with anhydrous ethanol, and the precipitate was collected by freeze drying to obtain MON; S2. Preparation of drug-loaded mesoporous organosilica nanoparticles: The MON obtained in the above steps was dispersed in water, APTES was added, magnetic stirring was performed, centrifugation was carried out, the supernatant was discarded, and the precipitate was washed with ultrapure water and collected to obtain the amino-modified MON, i.e., MON-NH2; Succinic acid was added to ethanol containing EDC and NHS for activation, and the succinic acid solution was added to the MON-NH2 prepared above, magnetic stirring was performed, centrifugation was carried out, and the precipitate was collected. The precipitate was washed with purified water and ethanol in sequence and collected to obtain the carboxyl-modified MON, i.e., MON-COOH; DOX / Que ethanol solution was added to the MON-COOH prepared above, stirred in the dark, centrifuged, washed with ethanol, and the precipitate was collected to obtain the MON nanoparticles DQM co-loaded with DOX and Que; S3. Preparation of drug-loaded mesoporous organosilicon nanoparticles coated with metal polyphenol network: The prepared DQM nanoparticles were ultrasonically dispersed and dissolved in purified water. CuCl2 solution was added, and the mixture was stirred in the dark. Tannic acid solution was added, and the mixture was stirred in the dark. After centrifugation, the nanoparticles were washed with purified water to obtain MPN-coated DQM nanoparticles formed by Cu and TA. These nanoparticles were then dispersed in hyaluronic acid solution, stirred in the dark, centrifuged, and washed with purified water to obtain hyaluronic acid-modified DQMCT nanoparticles.

2. The method for preparing drug-loaded mesoporous organosilicon nanoparticles coated with a metal polyphenol network as described in claim 1, characterized in that: In step S2, the volume ratio of succinic acid to MON-NH2 is 1:1~20.

3. The method for preparing drug-loaded mesoporous organosilicon nanoparticles coated with a metal polyphenol network as described in claim 1, characterized in that: In step S2, the mass ratio of DOX / Que ethanol solution to MON-COOH is 0.5~1:1~20.

4. The method for preparing drug-loaded mesoporous organosilicon nanoparticles coated with a metal polyphenol network as described in claim 1, characterized in that: In step S2, the concentration of the DOX / Que ethanol solution is 200-600 μg / mL.

5. The method for preparing drug-loaded mesoporous organosilicon nanoparticles coated with a metal polyphenol network as described in claim 1, characterized in that: In step S3, the mass ratio of TA to Cu is 1:1, and the concentration of TA is 1.2~60.0 mM.

6. The method for preparing drug-loaded mesoporous organosilicon nanoparticles coated with a metal polyphenol network as described in claim 1, characterized in that: In step S3, the concentration of hyaluronic acid is 0.1~2.0 mg / mL.

7. Hyaluronic acid-modified DQMCT nanoparticles prepared by the method according to any one of claims 1-6.

8. The use of the hyaluronic acid-modified DQMCT nanoparticles as described in claim 7 in the preparation of drugs for treating drug-resistant breast cancer.

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