Silicon dioxide-based nano-targeted drug delivery system for synergistic treatment of breast cancer

Abstract

The present application belongs to the technical field of tumor treatment, and specifically discloses a copper silicate-based nano-targeted drug delivery system for synergistically treating breast cancer, which comprises CuSiO3, tamoxifen, narcissus-derived carbon quantum dots, polyethylene glycol and hyaluronic acid; tamoxifen and narcissus-derived carbon quantum dots are loaded on CuSiO3 through electrostatic interaction, and then polyethylene glycol is covalently modified, and then hyaluronic acid with targeting ability is coated to prepare a CuSiO3@TAF@CDs-PEG-HA nano-targeted drug delivery system. The copper silicate-based nano-targeted drug delivery system for synergistically treating breast cancer is used, and based on the imbalance of intracellular redox homeostasis caused by oxidative stress and weakened antioxidant capacity, the copper silicate-based nano-targeted drug delivery system exhibits high tumor treatment effect on human breast cancer cells and tumor-bearing mouse models.

Description

Technical Field

[0001] The present invention relates to the technical field of tumor treatment, in particular to a copper silicate-based nano-targeted drug-carrying system for synergistically treating breast cancer. Background Art

[0002] Although chemodynamic therapy (CDT) has shown great potential as a broad therapeutic strategy based on reactive oxygen species (ROS) in cancer treatment, its practical application still faces many challenges. Therefore, further improving the efficiency of the reaction to increase the production of cytotoxic hydroxyl radicals (·OH) is a key issue that needs to be addressed.

[0003] Maintaining redox balance is a prerequisite for the survival and growth of cancer cells. In order to maintain redox balance, cancer cells enhance their antioxidant capacity by producing more reduced glutathione (GSH) to cope with the oxidative damage caused by excessive reactive oxygen species (ROS). In view of this, one strategy to kill cancer cells is usually to destroy their redox homeostasis, on the one hand, to produce higher levels of reactive oxygen species (ROS) and enhance the oxidative stress of the cells, and on the other hand, to weaken the antioxidant capacity of the cells and make their defense function deficient. However, most current research focuses on one aspect, and few therapeutic drugs can achieve both goals at the same time. A nano-copper-based metal-organic framework (CuHPT) was prepared by self-assembly of copper ions and catechol ligands (HPT), which overcomes cancer chemoresistance by destroying cellular redox homeostasis. CuHPT oxidizes endogenous GSH into oxidized glutathione (GSSG), effectively consuming GSH, and at the same time, the CuHPT structure decomposes and releases Cu. + and HPT, Cu + Catalyzes H2O2 to generate hydroxyl radicals (·OH), and HPT promotes O2 through electronic oxidation ·- CuHPT's unique metal-organic framework structure and ROS amplification effect effectively inhibit the growth of drug-resistant colorectal tumors and double the survival time of tumor-bearing mice.

[0004] Carbon quantum dots (CDs) are highly dispersed carbon nanoparticles with a size below 10 nm. CDs possess unique photoluminescence properties (dependent on size and excitation wavelength), low cytotoxicity, and excellent thermal stability. Therefore, they hold broad application prospects in research areas such as bioimaging, free radical scavenging, analytical sensing, and disease diagnosis and treatment.

[0005] In recent years, biomass-derived CDs have attracted extensive attention due to their excellent properties, including low cost, rich surface functional groups, environmental friendliness, biocompatibility, and the like. Recent studies have shown that biomass-derived CDs can mimic the structure and function of natural enzymes as nanoenzymes, especially GSH oxidase, catalase or superoxide dismutase. This unique enzyme-like property provides a new and effective way for oxidative stress-related research and intervention in biological systems. Coffee-derived chlorogenic acid (ChA) CDs were prepared by a simple hydrothermal reaction, which can generate GSSG by reducing GSH and exhibit significant GSH oxidase-like activity. Through promoting GSH depletion and imbalance of redox reactions caused by inactivation of glutathione peroxidase 4 (GPX4), iron death of human hepatoma cells (HepG2) cells is induced.

[0006] Inspired by the above research, in order to solve the above problems, the present application proposes a strategy to enhance oxidative stress of cancer cells while weakening the antioxidant system, which breaks the redox homeostasis in cancer cells and induces cell death. First, the anti-estrogen drug tamoxifen (TAF) and narcissus-derived carbon quantum dots (CDs) are loaded on CuSiO3 by electrostatic interaction, and then polyethylene glycol (PEG) is covalently modified on the surface of CuSiO3@TAF@CDs to improve the dispersion stability in solution to obtain CuSiO3@TAF@CDs-PEG (CTCP), and finally the CuSiO3@TAF@CDs-PEG (CTCP) surface is coated with hyaluronic acid (HA) with targeting ability to design and prepare CuSiO3@TAF@CDs-PEG-HA (CTCPH) nano-targeted drug delivery system. SUMMARY

[0007] The purpose of the present application is to provide a copper silicate-based nano-targeted drug delivery system for synergistic treatment of breast cancer, which is used for the synergistic treatment of breast cancer to destroy the redox homeostasis. Based on the imbalance of intracellular redox homeostasis caused by oxidative stress and weakened antioxidant capacity, the copper silicate-based nano-targeted drug delivery system exhibits high tumor treatment effect on human breast cancer cells and tumor-bearing mouse models.

[0008] To achieve the above objectives, the present invention provides a copper silicate-based nano-targeted drug delivery system for the synergistic treatment of breast cancer, comprising CuSiO3, tamoxifen, narcissus-derived carbon quantum dots, polyethylene glycol, and hyaluronic acid; tamoxifen and narcissus-derived carbon quantum dots are loaded on CuSiO3 by electrostatic action to generate CuSiO3@TAF@CDs, and then the surface of CuSiO3@TAF@CDs is covalently modified with polyethylene glycol to obtain CuSiO3@TAF@CDs-PEG, and hyaluronic acid with targeting ability is coated on the surface of CuSiO3@TAF@CDs-PEG to prepare a CuSiO3@TAF@CDs-PEG-HA nano-targeted drug delivery system.

[0009] Preferably, the loading concentration of tamoxifen is 1 mg / mL.

[0010] Preferably, the preparation method of the CuSiO3 is as follows:

[0011] A. Synthesis of Cu2O: Dissolve Cu(NO3)2 and NH4NO3 in deionized water, add NaOH solution at a rate of 1.8 mL / min, continue stirring, add ascorbic acid solution at a rate of 0.6 mL / min, stir again, collect the product Cu2O by centrifugation, wash with ethanol, and disperse in ethanol;

[0012] B. Synthesis of Cu2O@mSiO2: The Cu2O dispersion was diluted in a mixture of ethanol and deionized water. A 25 wt% aqueous solution of hexadecyltrimethylammonium chloride and triethylamine diluted 10-fold with ethanol were added in sequence. After adding ethyl orthosilicate, the mixture was stirred at room temperature. The solid Cu2O@mSiO2 was collected by centrifugation and washing, and then redispersed in ethanol.

[0013] C. Synthesis of CuSiO3: 2-Methylimidazole and ethanol were added to the Cu2O@mSiO2 dispersion, stirred at room temperature, centrifuged and washed twice with ethanol to obtain CuSiO3, which was redispersed in ethanol for later use.

[0014] Preferably, the preparation method of narcissus-derived carbon quantum dots is as follows:

[0015] Weigh narcissus bulb powder and put it into a crucible, air-roast it at 200℃ for 2h, and after cooling, prepare it according to the mass ratio of carbonized narcissus powder and pure water of 1:100, and then ultrasonicate it at room temperature for 1h. After the ultrasonication, collect the supernatant by centrifugation and filter it with a microporous filter membrane with a pore size of 0.22μm to obtain a brown clear solution, which is then dialyzed for purification and finally dried in a freeze dryer to obtain narcissus-derived carbon quantum dot powder.

[0016] Preferably, the preparation method of CuSiO3@TAF@CDs is as follows:

[0017] Tamoxifen powder and an aqueous solution of narcissus-derived carbon quantum dots were added to the CuSiO3 ethanol dispersion at a mass ratio of CuSiO3:tamoxifen:narcissus-derived carbon quantum dots of 2:1:1. The mixture was ultrasonically dispersed evenly, stirred at room temperature overnight, and the precipitate was collected by centrifugation to obtain CuSiO3@TAF@CDs.

[0018] Preferably, the preparation method of CuSiO3@TAF@CDs-PEG is as follows:

[0019] CuSiO3@TAF@CDs was dispersed in ethanol, and silane polyethylene glycol was added at a CuSiO3:polyethylene glycol mass ratio of 1:5 and ultrasonically dispersed uniformly. The mixture was reacted at 60°C for 24 h, and the precipitate was collected by centrifugation to obtain CuSiO3@TAF@CDs-PEG.

[0020] Preferably, the preparation method of CuSiO3@TAF@CDs-PEG-HA is as follows:

[0021] CuSiO3@TAF@CDs-PEG was dispersed in ethanol, and an aqueous solution of hyaluronic acid was added at a CuSiO3:hyaluronic acid mass ratio of 2:1. The mixture was stirred at room temperature overnight, and the precipitate was collected by centrifugation to obtain CuSiO3@TAF@CDs-PEG-HA.

[0022] The advantages and beneficial effects of the present invention using the above-mentioned copper silicate-based nano-targeted drug delivery system for synergistic treatment of breast cancer are:

[0023] 1. The present invention develops a CuSiO3@TAF@CDs-PEG-HA (CTCPH) nano-targeted drug delivery system for synergistic treatment of breast cancer that disrupts redox homeostasis. By loading the drug TAF on CuSiO3 with a drug loading rate of 30.8%, the lactic acid content is increased to reduce the intracellular pH, thereby enhancing the efficacy of CDT and cellular oxidative stress; at the same time, CDs with GSH oxidase activity are loaded to promote GSH depletion and weaken the antioxidant system of cancer cells. Finally, PEG is covalently modified on the surface as a tumor targeting agent and HA is introduced. The targeted CTCPH enters the human breast cancer cells and undergoes acidic degradation within 24 hours, cumulatively releasing 83.7% of TAF and 49.3% of Cu. 2+ , the Cu ion-mediated Fenton-like reaction promotes ROS production, and TAF-mediated reduction of intracellular pH can enhance ROS generation. In addition, the released CDs act as GSH oxidizing nanozymes to promote the consumption of antioxidant GSH.

[0024] 2. This invention exploits the imbalance in intracellular redox homeostasis caused by oxidative stress and impaired antioxidant capacity, resulting in CTCPH demonstrating a highly effective tumor treatment effect in both human breast cancer cells and tumor-bearing mouse models, with a tumor inhibition rate of 76.4%. H&E staining of major organs also demonstrated CTCPH's robust biosafety, making it a promising innovative strategy for precision cancer treatment.

[0025] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The synthesis and characterization of CDs of the present invention are shown in Figure 1, wherein (a) is a transmission electron microscopy image of CDs, (b) is a particle size distribution diagram of CDs, (c) is an X-ray diffraction spectrum of CDs, (d) is an AFM image and thickness distribution of CDs, and (e) is a three-dimensional AFM image of CDs.

[0027] Figure 2 Spectra of CDs of the present invention, wherein (a) is the ultraviolet absorption spectrum of CDs, and (b) is the excitation and emission spectra of CDs;

[0028] Figure 3 1 is a characterization spectrum of CTCPH of the present invention, wherein (a) is a TEM image of CTCPH, (b) is an XRD pattern of CTCPH, (c) is an FTIR spectrum of CTCPH, and (d) is a Zeta potential diagram of CTCPH;

[0029] Figure 4 The UV-vis spectra of TAF at different concentrations and the corresponding standard curves of the present invention are shown, wherein (a) is the UV-vis spectra of TAF at different concentrations, and (b) is the standard curve corresponding to TAF;

[0030] Figure 5 1 is a graph showing the results of an in vitro acid-responsive release test of the present invention, wherein (a) is a TEM image of CTCPH after immersion at pH 5.4 for 24 hours, (b) is a partially enlarged image of (a), (c) is a cumulative release curve of TAF from CTCPH at different pH values, and (d) is a cumulative release curve of Cu ions from CTCPH at different pH values;

[0031] Figure 6 Graphs showing the results of GSH consumption and ·OH testing of the present invention, wherein (a) is a diagram showing the mechanism of GSH oxidase-like activity of CDs, (b) is a diagram showing the change in absorbance over time when CDs and GSH are added, (c) is a Michaelis-Menten curve, and (d) is a double reciprocal graph;

[0032] Figure 7The results of the consumption of GSH by CTCPH and the generation of ·OH by CTCPH under different conditions are shown in the figure, wherein (a) is the consumption of GSH in the presence of CTPH or CTCPH, (b) is the consumption of GSH under different concentrations of CTCPH, (c) is a diagram showing the mechanism of GSH consumption and ROS generation by CTCPH, (d) is the degradation of MB after treatment with CTCPH at different pH conditions, (e) is the degradation of MB after treatment with CTCPH for different time periods at pH = 5.4, and (f) is the ESR spectra corresponding to different pH conditions under CTCPH treatment;

[0033] Figure 8 2 are graphs showing the results of intracellular pH and ROS level detection of the present invention, wherein (a) is the relative activity of mitochondrial complex I in MCF-7 cells treated with PBS (control group) and CTCPH for 12 h, (b) is the intracellular lactate level in MCF-7 cells treated with PBS (control group) and CTCPH for 12 h, (c) is a CLSM image of MCF-7 cells stained with BCECF-AM after different treatments, and (d) is a CLSM image of MCF-7 cells stained with DCFH-DA after different treatments;

[0034] Figure 9 Figure 1 is a graph showing the cytotoxicity of CTCPH tested by the CCK-8 assay of the present invention, wherein (a) is the activity evaluation of MCF-7 cells, (b) is the activity evaluation of HUVEC cells; (c) is the CLSM image of MCF-7 cells stained with Calcein-AM and PI under different treatment conditions;

[0035] Figure 10 is a CLSM image of MCF-7 cells of the present invention after incubation with CTCPH for 6 h;

[0036] Figure 11 The figures are the in vivo anti-tumor efficacy and biosafety evaluation results of the present invention, wherein (a) shows the establishment of the MCF-7 tumor model and the treatment plan, (b) shows representative photos of tumors in different groups after 14 days of treatment, (c) shows the curves of tumor volume changes in different groups during treatment, and (d) shows the curves of weight changes in mice in different groups during treatment;

[0037] Figure 12 These are H&E staining and TUNEL staining images of tumor sections in different treatment groups of the present invention. DETAILED DESCRIPTION

[0038] The technical solution of the present invention is further described below with reference to the accompanying drawings and embodiments.

[0039] Unless otherwise defined, technical or scientific terms used in the present invention shall have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.

[0040] Unless otherwise defined, the reagents, equipment and other materials used in the present invention are all commercially available.

[0041] Example 1

[0042] A copper silicate-based nano-targeted drug delivery system for the synergistic treatment of breast cancer includes CuSiO3, tamoxifen (TAF), narcissus-derived carbon quantum dots (CDs), polyethylene glycol (PEG), and hyaluronic acid (HA); tamoxifen (TAF) and narcissus-derived carbon quantum dots (CDs) are loaded on CuSiO3 via electrostatic interaction to generate CuSiO3@TAF@CDs, and then the surface of CuSiO3@TAF@CDs is covalently modified with polyethylene glycol (PEG) to obtain CuSiO3@TAF@CDs-PEG (CTCP), and the surface of CuSiO3@TAF@CDs-PEG (CTCP) is coated with hyaluronic acid (HA) with targeting ability to prepare the CuSiO3@TAF@CDs-PEG-HA (CTCPH) nano-targeted drug delivery system.

[0043] The loading concentration of tamoxifen (TAF) was 1 mg / mL.

[0044] The preparation method of CuSiO3 is as follows:

[0045] A. Synthesis of Cu2O: Synthesis of Cu2O: Dissolve 0.1 mmol of Cu(NO3)2 and 0.12 mmol of NH4NO3 in 40 mL of deionized water. Then, at a specific speed of 1.8 mL min -1 Add 2.0 mL of a 0.2 M NaOH solution. Stirring continues for 10 minutes, then add 3.5 mL of a 0.1 M ascorbic acid solution at a specific rate of 0.6 mL / min. Stir for an additional 10 minutes. After the reaction is complete, the Cu2O product is collected by centrifugation, washed with ethanol, and dispersed in 10 mL of ethanol.

[0046] B. Synthesis of Cu2O@mSiO2: The above Cu2O dispersion was diluted in a mixture of 20 mL of ethanol and 10 mL of deionized water. Subsequently, 0.16 mL of a 25 wt% aqueous solution of hexadecyltrimethylammonium chloride (CTAC) and 0.15 mL of triethylamine (TEA) diluted 10-fold with ethanol were added in sequence. After adding 50 μL of tetraethyl orthosilicate (TEOS), the mixture was stirred at room temperature for 14 h. The solid Cu2O@mSiO2 was collected by centrifugation and washing and redispersed in 10 mL of ethanol.

[0047] C. Synthesis of CuSiO3: Add 2 mL of 0.2 g / mL 2-methylimidazole (2-Melm) and 4.5 mL of ethanol to 1 mL of the Cu2O@mSiO2 dispersion. Stir at room temperature for 14 h. After centrifugation and washing twice with ethanol, the resulting CuSiO3 was redispersed in 10 mL of ethanol for later use.

[0048] CuSiO3 is a layered CuSiO3 nanoflower carrier.

[0049] Cu2O nanospheres were synthesized via a room-temperature ascorbic acid reduction method. During the synthesis, NH4NO3 was added to inhibit the rapid growth of the Cu(OH)2 intermediate phase and enable the size-controlled growth of the Cu2O crystals. A Cu2O@mSiO2 core-shell structure was formed by uniformly growing a mesoporous SiO2 shell on the Cu2O core. When the 2-MeIm content reached 0.2 g / mL, the core-shell Cu2O@mSiO2 structure completely transformed into a uniform nanoflower-like structure (i.e., CuSiO3). This phenomenon indicates that 2-MeIm plays a key role in mediating the phase transition from the core-shell Cu2O@mSiO2 structure to the hollow CuSiO3 structure.

[0050] The preparation method of narcissus-derived carbon quantum dots is as follows:

[0051] Weigh narcissus bulb powder and put it into a crucible, air-roast it at 200℃ for 2h, and after cooling, prepare it according to the mass ratio of carbonized narcissus powder and pure water of 1:100, and then ultrasonicate it at room temperature for 1h. After the ultrasonication, collect the supernatant by centrifugation and filter it with a microporous filter membrane with a pore size of 0.22μm to obtain a brown clear solution, which is then dialyzed for purification and finally dried in a freeze dryer to obtain narcissus-derived carbon quantum dots (CDs) powder.

[0052] The preparation method of CuSiO3@TAF@CDs is as follows:

[0053] Tamoxifen powder and an aqueous solution of narcissus-derived carbon quantum dots were added to the CuSiO3 ethanol dispersion at a mass ratio of CuSiO3:tamoxifen:narcissus-derived carbon quantum dots of 2:1:1. The mixture was ultrasonically dispersed evenly, stirred at room temperature overnight, and the precipitate was collected by centrifugation to obtain CuSiO3@TAF@CDs.

[0054] The preparation method of CuSiO3@TAF@CDs-PEG (CTCP) is as follows:

[0055] CuSiO3@TAF@CDs was dispersed in ethanol, and silane polyethylene glycol was added at a CuSiO3:polyethylene glycol mass ratio of 1:5 and ultrasonically dispersed uniformly. The mixture was reacted at 60°C for 24 h, and the precipitate was collected by centrifugation to obtain CuSiO3@TAF@CDs-PEG.

[0056] The preparation method of CuSiO3@TAF@CDs-PEG-HA(CTCPH) is as follows:

[0057] CuSiO3@TAF@CDs-PEG was dispersed in ethanol, and an aqueous solution of hyaluronic acid was added at a CuSiO3:hyaluronic acid mass ratio of 2:1. The mixture was stirred at room temperature overnight, and the precipitate was collected by centrifugation to obtain CuSiO3@TAF@CDs-PEG-HA.

[0058] For CuSiO3@CDs-PEG-HA (CCPH) and CuSiO3@TAFs-PEG-HA (CTPH), the preparation method was the same as above, in which the mass of CuSiO3 was kept the same.

[0059] Example 2

[0060] The performance of the synthesized CuSiO3@TAF@CDs-PEG-HA was verified.

[0061] 1. Synthesis and characterization of CTCPH.

[0062] CDs were obtained by a series of treatments using narcissus bulbs as precursors. First, the morphology and size distribution of CDs particles were studied using transmission electron microscopy (TEM). CDs were uniformly dispersed spherical nanoparticles with no obvious aggregation ( Figure 1 (a)). High resolution images show that these CDs have obvious lattice fringes with a lattice fringing spacing of 0.21 nm, corresponding to the (100) crystal plane of graphitic carbon. According to particle size distribution statistics, the average particle size of CDs is 2.4 ± 0.3 nm ( Figure 1 In order to further clarify its structural characteristics, X-ray diffraction (XRD) was used to analyze the crystal structure of CDs ( Figure 1 In (c), it can be found that CDs have a broad diffraction peak at 20°, which can be attributed to the diffraction surface of amorphous graphitic carbon, indicating the successful preparation of CDs. In addition, the surface morphology of CDs was further observed by atomic force microscopy (AFM). Figure 1 In (d) and (e), the images show that the CDs are evenly distributed and well dispersed, and their height is basically consistent with the average particle size, further indicating that they have a spherical or quasi-spherical structure.

[0063] The UV-visible spectrum of CDs is as follows Figure 2As shown in (a). CDs exhibit a wide range of UV-visible absorption between 200 and 550 nm, with strong light absorption capacity; the strong absorption band near 264 nm is attributed to the π-π* electronic transition in the C=C bond, which originates from the conjugated π orbital of the carbon ring and is related to the (crystal) carbon core. Figure 2 As shown in (b), CDs exhibit excitation-dependent fluorescence (photoluminescence, PL) in the 320-400 nm range, with optimal excitation at 364 nm and optimal emission at 439 nm. The inset shows photographs of a CDs aqueous solution and a pure water solution under 365 nm ultraviolet radiation. Compared to pure water, the CDs aqueous solution exhibits significant blue fluorescence under UV excitation. These results demonstrate the excellent fluorescence properties of CDs.

[0064] After TAF and CDs nanoparticles were fixed on CuSiO3 through electrostatic interaction, PEG was modified by covalent action and further coated with HA to prepare CuSiO3@TAF@CDs-PEG-HA (CTCPH) nano-targeted drug delivery system. A large number of evenly dispersed CDs nanoparticles can be seen in the TEM image. CuSiO3 still retains the complete morphology of nanoflowers and is not damaged by the loading and functionalization steps ( Figure 3 (a)). XRD pattern of CTCPH ( Figure 3 (b)) has no significant change compared to CuSiO3, indicating that the basic crystal structure of CuSiO3 is not affected by loading and functional modification. Fourier transform infrared (FTIR) spectrum ( Figure 3 (c) shows that at 1040cm -1 The characteristic peak of C=O appeared at 3132 cm -1 The absorption peak of CuSiO3 is considered to be the stretching vibration of -CH2- in PEG, which further proves the successful PEGylation of CuSiO3. In addition, the change of Zeta potential further proves the successful preparation of CTCPH nanocomposite system ( Figure 3 (d)).

[0065] 2. Drug loading capacity of CTCPH.

[0066] Figure 4The absorption curves of TAF solutions of different concentrations in the UV-Vis spectrum are shown. These curves correspond to the absorption of TAF solutions of different concentrations (4-60 μg / mL) in the wavelength range of 200-400 nm, where the characteristic UV peak of TAF is at 238 nm. The standard curve in the figure shows the linear relationship between TAF concentration and absorption value. The linear regression equation is y=0.03x+0.014, where x represents the concentration of TAF and y represents the absorbance. The R of the linear regression is 2 The value was 0.999, indicating a high linear correlation between concentration and absorbance. Using the TAF standard curve, the TAF concentration can be estimated in subsequent experiments based on the absorbance of the supernatant of the drug-loaded nanomaterials.

[0067] Drug loading rate and loading efficiency are two key indicators that describe the degree to which drugs are loaded in drug delivery systems. A high drug loading rate means that the carrier can carry more drugs, and a high loading efficiency means that the loading capacity of the carrier is effectively utilized. Ideally, both the drug loading rate and loading efficiency are as high as possible. Table 1 shows the TAF drug loading rate and loading efficiency when TAF is loaded using a CuSiO3 carrier. It contains five different TAF initial concentrations (1.0, 0.8, 0.4, 0.2, 0.1 mg / mL). From these data, it can be seen that the absorbance, drug loading rate and loading efficiency of TAF decrease as the initial concentration decreases. Taking all factors into consideration, the subsequent experiments selected a TAF loading concentration of 1 mg / mL for further analysis and application.

[0068] Table 1 Results of CuSiO3 loaded TAF

[0069] <![CDATA[TAF初始浓度(mgmL -1 )]]> Drug loading rate (%) Loading efficiency (%) 1.0 30.8 92.5 0.8 26.3 91.9 0.4 15.5 92.8 0.2 8.3 91.4 0.1 4.2 88.7

[0070] 3. In vitro acid response release test.

[0071] like Figure 5 As shown in the figure, similar to CuSiO3, CTCPH also exhibits acid-responsive degradation behavior. Under weak acidic conditions (pH = 5.4), after incubation for 24 hours, the nanoflower structure of CTCPH dissociates and significantly decomposes into fragments. The results further confirm that the nanomedicine has pH-dependent degradation characteristics. Subsequently, the release of TAF and copper ions from CTCPH under normal physiological conditions (pH = 7.4) and weak acidic conditions (pH = 5.4) was studied. Figure 5 As shown in Figure 3, CTCPH exhibited excellent chemical stability under normal physiological conditions, with only 14.7% of TAF and 7.4% of Cu ions released within 24 hours. In contrast, CTCPH exhibited a significant burst release pattern under weakly acidic conditions simulating tumors, with cumulative release of TAF and Cu within 24 hours. 2+Therefore, it can be speculated that CTCPH maintains its structural integrity when circulating in neutral blood and rapidly releases TAF and Cu ions in situ after entering weakly acidic tumor tissue, thereby effectively reducing the nonspecific release of TAF and Cu.

[0072] 4. GSH consumption and hydroxyl radical (·OH) detection.

[0073] Figure 6 (a) shows a schematic diagram of the GSH oxidase-like activity of CDs. Reduced glutathione (GSH) is converted into oxidized glutathione (GSSG) through the catalytic action of CDs, and 5,5'-dithio-2-nitrobenzoic acid (DTNB) reacts to generate a yellow product containing nitrite (TNB). Figure 6 (b) shows the change in absorbance at 412 nm over time at different concentrations of GSH (from 0.5 to 10 mM) in a system containing CDs (100 μg / mL) and DTNB (100 μM). As can be seen from the figure, as the concentration of GSH increases, the absorbance also increases, and as time goes by, the absorbance also decreases, indicating that GSH is effectively removed by CDs. Subsequently, quantitative analysis of enzyme kinetics showed that CDs exhibited typical Michaelis-Menten kinetics in the DTNB color reaction ( Figure 6 In addition, the V of CDs was determined by double reciprocal plot analysis. max and K m 4.3810-6M and 2.7210-7Mmin respectively -1 ( Figure 6 These results indicate that CDs exhibit GSH oxidase-like activity under the experimental conditions, and its kinetic characteristics are consistent with the classical Michaelis-Menten model.

[0074] Different concentrations of CTCPH (50, 100, and 150 μg / mL) were incubated with excess GSH, and the remaining GSH was then determined using a DTNB-containing solution. As the concentration increased, the specific absorbance at 412 nm decreased, indicating that CTCPH could effectively deplete GSH ( Figure 7 (a)), which is attributed to the release of a large amount of Cu 2+ It can oxidize GSH to GSSG. In addition, CTCPH showed enhanced GSH consumption ability compared with CTPH (CuSiO3@TAF-PEG-HA) without CDs loading ( Figure 7 (b)), which further confirms that the GSH oxidase-like activity of CDs can further enhance CTCPH-mediated GSH consumption ( Figure 7(c)).

[0075] The results of CTCPH producing ·OH under different conditions were investigated. The degradation of methylene blue (MB) under different pH conditions (7.4, 6.4, 5.4) under CTCPH treatment was as follows: Figure 7 As shown in (d), the absorbance of MB gradually decreases with the decrease of pH value, indicating that MB is gradually degraded and the amount of ·OH generated increases. Figure 7 Figure (e) shows the changes in the absorbance of MB at different time points (1, 6, 12, and 24 hours) under pH = 5.4. As the reaction time increases, the absorbance of MB gradually decreases, indicating that the amount of OH generated is positively correlated with time. In addition, free radicals were detected by ESR experiments. The ESR spectra of MB at different pH conditions under CTCPH treatment are shown in Figure 5. Figure 7 As shown in (f), as the pH value decreases, the peak intensity of ·OH gradually increases, indicating that the amount of ·OH generated increases, which is consistent with the previous results of MB degradation. The decrease in pH value increases the amount of ·OH generated. This phenomenon can be attributed to the increase in environmental acidity, which also promotes the formation of Cu in CTCPH. 2+ mediated Fenton-like reaction rate.

[0076] 5. Detection of intracellular pH and ROS levels.

[0077] Tamoxifen (TAF) is an anti-estrogen drug widely used in the treatment of estrogen receptor-positive (ER+) breast cancer. It can inhibit mitochondrial complex I located on the inner membrane of mitochondria. When the activity of mitochondrial complex I is inhibited, cell metabolism shifts from oxidative phosphorylation to glycolysis, leading to the accumulation of lactic acid. The accumulation of lactic acid will enhance the intracellular acidic environment, thereby enhancing the efficiency of Fenton-like catalysis and increasing the level of intracellular ROS. First, the activity of the mitochondrial complex I of the sample is detected. Complex I can catalyze the dehydrogenation of reduced nicotinamide adenine dinucleotide (NADH) to generate oxidized nicotinamide adenine dinucleotide (NAD+). The oxidation rate of NADH is measured at 340nm to calculate the activity of the enzyme. Figure 8 As shown in (a), compared with the control group without drug treatment, the activity of mitochondrial complex I in the CTCPH group was significantly inhibited, decreasing to about 0.2 times that of the control group. Then, the lactate content was detected using a lactate content detection kit. The detection principle is as follows: lactate generates pyruvate under the action of lactate dehydrogenase (LDH), and at the same time, NAD + Reduction to generate NADH and H + , H + Transferred to phenazine methyl sulfate (PMS), the generated PMSH2 reduces MTT to generate purple Formazan, which has a characteristic absorption peak at 570nm. Figure 8As shown in (b), compared with the untreated control cell group, the lactate content in the CTCPH-treated cells increased relatively, reaching approximately 1.8 times that of the control group.

[0078] To investigate the ability of CTCPH to increase the intracellular acidic environment, a pH fluorescent probe (BCECF-AM) was used to measure the intracellular pH value of human breast cancer cell line (MCF-7). Figure 8 In (c), BCECF-AM is highly sensitive to pH, with its green fluorescence intensity decreasing with increasing acidity. Confocal laser scanning microscopy (CLSM) images show that the green fluorescence signal of MCF-7 cells in the CTCPH group is significantly weaker than that in the blank control group and the CCPH group without TAF loading, demonstrating that CTCPH indeed induces increased intracellular acidity. A fluorescent probe (DCFH-DA) was further used to evaluate ROS generation in MCF-7 cells under different treatment conditions. Figure 8 As can be seen in (d), the ROS green fluorescence signal in the control group was almost negligible, indicating relatively low ROS levels, while significant fluorescence signals were observed in cells treated with CCPH and CTCPH. In particular, the CTCPH group showed the strongest ROS fluorescence signal, demonstrating enhanced ROS generation. These studies demonstrate that inhibition of complex I activity induced by TAF loaded in CTCPH can specifically lower intracellular pH, thereby overcoming the inherent acidity deficiency in tumors, improving the efficiency of the Cu ion-mediated Fenton-like reaction, and increasing ROS levels.

[0079] 6. Cytotoxicity detection and cellular uptake.

[0080] Given the enhanced GSH depletion and increased ROS generation induced by CTCPH, the cytotoxicity of CTCPH was further tested by CCK-8 assay ( Figure 9(a)). TAF, CuSiO3, and CTCP (CuSiO3@TAF@CDs-PEG) were co-cultured with CTCPH cells for 24 hours to explore their toxic effects on MCF-7 cells. The results showed that all treatments exhibited a dose-dependent inhibitory effect on MCF-7, and TAF and CuSiO3 carriers alone exhibited certain cytotoxicity, which was related to TAF-mediated chemotherapy and released Cu ion-mediated chemodynamic therapy. However, the CTCP group and CTCPH group exhibited higher cytotoxicity, which means that the loaded CDs and TAF played a synergistic role with the CuSiO3 carrier, especially in the CTCPH group. As the CTCPH concentration increased to 100 μg / mL, the cell survival rate of MCF-7 cells was less than 20%, which was the lowest compared to all other experimental groups. This was due to the modification of the HA targeting agent, which further increased the cellular uptake rate of CTCPH and increased the toxicity of MCF-7 cells. In addition, CTCPH had no obvious cytotoxicity to normal cells (HUVEC) ( Figure 9 In (b), cell viability remained above 90% at a treatment concentration of 100 μg / mL. This is due to the specific degradation and release of CTCPH in the tumor microenvironment, which results in less release of Cu ions and TAF in normal cells, and thus less ROS generation compared to MCF-7 cells. These results demonstrate that CTCPH has a selective therapeutic effect on MCF-7 cells.

[0081] In order to further reveal the cell killing performance under different treatments, a live cell / dead cell double staining kit (Calcein-AM / PI double staining kit) was used for co-staining experiments. Figure 9 As shown in (c), obvious red fluorescence signals and sparse green fluorescence signals were observed in the CTCPH group, while the TAF group and CuSiO3 group maintained a relatively high level of viable cells, as reflected in brightly clustered green fluorescence signals and weak red fluorescence signals. This further indicates that CTCPH has a strong ability to kill cancer cells. In addition, after 6 hours of CTCPH incubation, obvious blue fluorescence appeared in MCF-7 cells, which was due to the photoluminescence properties of CDs, indicating that CTCPH can be effectively taken up by MCF-7 ( Figure 10 ).

[0082] 7. CTCPH-mediated synergistic cell death mechanism.

[0083] The mechanism of action of CTCPH can be summarized as follows: (1) After CTCPH is taken up by MCF-7 cells through targeted action, the acidic microenvironment promotes the degradation of CTCPH, thereby releasing TAF, CDs and Cu ions from CTCPH; (2) the released Cu 2+Reacts with high levels of GSH in cells to generate Cu + and GSSG, the antioxidant GSH was consumed in large quantities, and Cu + It reacts with H2O2 in a Fenton-like reaction to generate cytotoxic ·OH; (3) the released CDs act as GSH oxidase to further enhance the oxidation of GSH to GSSG, promoting further depletion of intracellular GSH; (4) the released TAF can not only act as an anti-estrogen drug to kill cancer cells, but also act as an inhibitor of mitochondrial complex I to enhance lactate levels, leading to increased acidification in tumor cells and accelerating Cu + The Fenton-like reaction with H2O2 generates more ·OH within tumor cells, increasing their oxidative stress. Through this mechanism, CTCPH can leverage the synergistic effects of its components to disrupt intracellular redox homeostasis, thereby killing cancer cells.

[0084] 8. In vivo anti-tumor effect and biosafety evaluation.

[0085] In order to systematically evaluate the therapeutic effect of CTCPH, an MCF-7 nude mouse tumor model was first established, and the following substances were injected into the tail vein: phosphate buffered saline (PBS), TAF, CuSiO3, CTCP, and CTCPH ( Figure 11 (a)). During the 14-day treatment period, the tumor size of each group of mice was measured every other day to determine the tumor growth inhibition effect. Figure 11 As shown in (b), the CuSiO3 or TAF treatment groups partially inhibited tumor growth compared with the PBS control group. It is worth noting that the tumor-bearing mice treated with CTCPH achieved the best therapeutic effect compared with the CuSiO3 or TAF injection groups, which was due to the synergistic therapeutic effect caused by the redox homeostasis imbalance caused by enhanced ROS and GSH consumption and the HA targeting effect. From the tumor growth curve ( Figure 11 As shown in (c), compared to the rapidly growing PBS control group, free TAF exhibited limited drug effect due to low tumor accumulation, while the CTCP or CTCPH groups exhibited significant tumor growth inhibition. In particular, CTCPH injection exhibited a stronger growth inhibition effect on tumors due to the HA targeting effect, with a tumor inhibition rate of 76.4%, further confirming the excellent anti-tumor efficacy of CTCPH.

[0086] During the entire treatment period, the body weight of MCF-7 tumor-bearing mice in all groups increased to a certain extent ( Figure 11(d)), which shows that all treatments and doses have almost no adverse effects on the normal growth of mice. Subsequently, H&E staining and TUNEL staining of tumor tissues collected from different treatment groups confirmed that CTCPH has a significant tumor therapeutic effect ( Figure 12 ). As expected, the CTCPH group showed the most severe histological damage among all groups in the H&E and TUNEL staining experiments. In addition, no obvious pathological abnormalities were observed in the major organs of each group stained with H&E, which shows that the injected CTCPH has almost no toxic side effects on mice, has reliable biosafety, and is expected to become a candidate drug for precision tumor diagnosis and treatment. To evaluate blood safety, blood was collected from normal nude mice after injection of CTCPH and PBS, and blood biochemistry and complete blood cell analysis were performed to detect a series of key parameters. As can be seen from Table 2, compared with the control group, the main indicators of blood biochemistry and blood routine of mice receiving CTCPH did not show significant changes. The above results show that CTCPH has reliable biosafety and is expected to become a candidate drug for precision tumor treatment.

[0087] Table 2 Blood biochemistry and complete blood cell analysis

[0088] Untreated control CTCPH Reference range <![CDATA[WBC(10 9 / L)]]> 4.9±0.8 6.8±1.1 0.8～10.6 <![CDATA[RBC(10 12 / L)]]> 8.03±0.94 8.26±1.11 6.5～11.5 HGB (g / L) 142.7±5.4 122.6±4.8 110～165 HCT (%) 46.2±3.7 38.7±2.6 35～55 MCV (fL) 52.2±1.5 52.4±1.1 41～55 MCH (pg) 15.8±1.0 14.3±0.8 13～18 RDW (%) 13.7±0.8 13.6±0.9 12～19 <![CDATA[PLT(10 9 / L)]]> 714.3±35.8 655.7±40.3 400～1600 PDW 16.5±0.6 16.6±0.5 12～17.5 ALT (U / L) 68.06±2.24 74.56±2.70 10.06～96.47 AST (U / L) 139.62±17.19 189.80±14.35 36.31～235.48 ALB (g / L) 31.04±1.07 28.79±1.48 21.22～39.15 ALP (U / L) 181.81±3.09 201.26±3.70 22.52～474.35 BUN (mg / dL) 15.95±1.06 18.09±0.73 10.81～34.74 CREA (pmol / L) 15.20±1.05 13.88±1.25 10.91～85.09

[0089] In the present invention, narcissus bulbs are first used as precursors, and carbon dots (CDs) are obtained by high-temperature heat treatment. CDs were observed by TEM to be uniformly dispersed spherical nanoparticles with an average particle size of 2.4±0.3nm and obvious lattice fringes. The fluorescence spectrum shows that CDs have good fluorescence properties. Through electrostatic interaction and covalent modification, TAF, CDs, PEG and HA were loaded and modified on the CuSiO3 surface, and the CuSiO3@TAF@CDs-PEG-HA (CTCPH) nano-targeted drug delivery system was successfully prepared. TEM, XRD and FTIR analyses confirmed the successful preparation and functional modification of CTCPH. The loading capacity of CTCPH for the drug TAF was determined by UV-vis spectroscopy and standard curve, and 1 mg mL -1 Subsequent experiments were conducted with a TAF concentration of 30.8%, and the drug loading rate and drug loading efficiency were 92.5%. In vitro acid-responsive release tests showed that CTCPH exhibited a significant burst release pattern under weakly acidic conditions (pH = 5.4), with a cumulative release of 83.7% of TAF and 49.3% of Cu within 24 hours. 2+ , indicating pH-dependent degradation. Furthermore, the GSH oxidase-like activity of CDs was verified, demonstrating their ability to effectively consume GSH. Furthermore, CTCPH was tested for its enhanced GSH consumption and enhanced ·OH generation under weakly acidic conditions.

[0090] Cell experiments showed that CTCPH further improved the efficiency of the Fenton-like reaction by inhibiting the activity of mitochondrial complex I (reduced to 0.2 times that of the control group) and enhancing the intracellular lactate content (increased to 1.8 times that of the control group). Cytotoxicity assay showed that CTCPH showed significant cytotoxicity to MCF-7 cells (100 μg mL -1 The cell survival rate was less than 20% when CTCPH was added to MCF-7 cells, while the toxicity to normal cells (HUVEC) was low (the cell survival rate remained above 90%). Cell uptake experiments showed that CTCPH could be effectively taken up by MCF-7 cells. The main mechanism of CTCPH-mediated synergistic cell death is as follows: CTCPH is degraded in MCF-7 cells to release TAF, CDs and Cu 2+ , Cu 2+ Consumes GSH to reduce to Cu + , Cu + CTCPH catalyzes H₂O₂ to generate ·OH. Besides its endocrine therapeutic effects, TAF can also lower intracellular pH, further promoting ·OH production. CDs, acting as GSH oxidases, enhance GSH consumption. The increased ROS and decreased GSH increase intracellular oxidative stress, thereby achieving a synergistic killing effect on MCF-7 cells. In vivo experiments showed that CTCPH exhibited significant tumor growth inhibition in an MCF-7 nude mouse tumor model, with a tumor inhibition rate of 76.4%, and exhibited minimal toxic side effects, demonstrating reliable biosafety. Blood biochemistry and complete blood cell analysis further confirmed the biosafety of CTCPH, indicating its potential as a candidate for precision cancer therapy.

[0091] Therefore, the present invention utilizes this copper silicate-based nanoparticle targeted drug delivery system for synergistic breast cancer treatment, specifically targeting redox homeostasis. Due to the imbalance in intracellular redox homeostasis caused by oxidative stress and weakened antioxidant capacity, the copper silicate-based nanoparticle targeted drug delivery system demonstrated highly effective tumor treatment in both human breast cancer cells and tumor-bearing mouse models.

[0092] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the same. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solutions of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A copper silicate-based nano-targeted drug delivery system for synergistic treatment of breast cancer, characterized by: The method comprises CuSiO3, tamoxifen, carbon quantum dots derived from narcissus, polyethylene glycol, and hyaluronic acid; tamoxifen and carbon quantum dots derived from narcissus are loaded on CuSiO3 via electrostatic interaction to generate CuSiO3@TAF@CDs, which is then covalently modified with polyethylene glycol to obtain CuSiO3@TAF@CDs-PEG, and hyaluronic acid with targeting ability is coated on the surface of CuSiO3@TAF@CDs-PEG to prepare a CuSiO3@TAF@CDs-PEG-HA nano-targeted drug delivery system; The loading concentration of tamoxifen was 1 mg / mL; The preparation method of narcissus-derived carbon quantum dots is as follows: Weigh narcissus bulb powder and put it into a crucible, air-roast it at 200℃ for 2h, and after cooling, prepare it according to the mass ratio of carbonized narcissus powder and pure water of 1:100, and then ultrasonicate it at room temperature for 1h. After the ultrasonication, collect the supernatant by centrifugation and filter it with a microporous filter membrane with a pore size of 0.22μm to obtain a brown clear solution, which is then dialyzed for purification and finally dried in a freeze dryer to obtain narcissus-derived carbon quantum dot powder.

2. The copper silicate-based nano-targeted drug delivery system for synergistic treatment of breast cancer according to claim 1, characterized in that: The preparation method of the CuSiO3 is as follows: A. Synthesis of Cu2O: Dissolve Cu(NO3)2 and NH4NO3 in deionized water, add NaOH solution at a rate of 1.8 mL / min, continue stirring, add ascorbic acid solution at a rate of 0.6 mL / min, stir again, collect the product Cu2O by centrifugation, wash with ethanol, and disperse in ethanol; B. Cu2O@ m Synthesis of SiO2: The above Cu2O dispersion was diluted in a mixture of ethanol and deionized water. 25 wt% aqueous hexadecyltrimethylammonium chloride solution and triethylamine diluted 10 times with ethanol were added in sequence. After adding ethyl orthosilicate, the mixture was stirred at room temperature. Solid Cu2O@ was collected by centrifugation and washing. m SiO2 and redispersed in ethanol; C. Synthesis of CuSiO3: 2-methylimidazole and ethanol were added to Cu2O@ m The SiO2 dispersion was stirred at room temperature, centrifuged and washed twice with ethanol to obtain CuSiO3, which was redispersed in ethanol for later use.

3. The copper silicate-based nano-targeted drug delivery system for synergistic treatment of breast cancer according to claim 1, characterized in that: The preparation method of CuSiO3@TAF@CDs is as follows: Tamoxifen powder and an aqueous solution of narcissus-derived carbon quantum dots were added to the CuSiO3 ethanol dispersion at a mass ratio of CuSiO3:tamoxifen:narcissus-derived carbon quantum dots of 2:1:

1. The mixture was ultrasonically dispersed evenly, stirred at room temperature overnight, and the precipitate was collected by centrifugation to obtain CuSiO3@TAF@CDs.

4. The copper silicate-based nano-targeted drug delivery system for synergistic treatment of breast cancer according to claim 1, characterized in that: The preparation method of CuSiO3@TAF@CDs-PEG is as follows: CuSiO3@TAF@CDs was dispersed in ethanol, and silane polyethylene glycol was added at a CuSiO3:polyethylene glycol mass ratio of 1:5 and ultrasonically dispersed uniformly. The mixture was reacted at 60°C for 24 h, and the precipitate was collected by centrifugation to obtain CuSiO3@TAF@CDs-PEG.

5. The copper silicate-based nano-targeted drug delivery system for synergistic treatment of breast cancer according to claim 1, characterized in that: The preparation method of CuSiO3@TAF@CDs-PEG-HA is as follows: CuSiO3@TAF@CDs-PEG was dispersed in ethanol, and an aqueous solution of hyaluronic acid was added at a CuSiO3:hyaluronic acid mass ratio of 2:

1. The mixture was stirred at room temperature overnight, and the precipitate was collected by centrifugation to obtain CuSiO3@TAF@CDs-PEG-HA.

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