Copper silicate-based nano-targeting drug delivery system for synergistically treating breast cancer

Abstract

The invention belongs to the technical field of tumor treatment, and particularly discloses a copper silicate-based nano targeting drug delivery system for synergistically treating breast cancer, which comprises CuSiO3, tamoxifen, narcissus-derived carbon quantum dots, polyethylene glycol and hyaluronic acid. The preparation method comprises the following steps: loading tamoxifen and narcissus-derived carbon quantum dots on CuSiO3 through electrostatic interaction, carrying out polyethylene glycol covalent modification, and coating with hyaluronic acid with targeting ability to prepare the CuSiO3 (at) TAF (at) CDs-PEG-HA nano targeting drug delivery system. According to the copper silicate-based nano-targeting drug delivery system for synergistically treating breast cancer, intracellular redox steady-state imbalance is caused based on oxidative stress and weakened antioxidant capacity, so that the copper silicate-based nano-targeting drug delivery system shows an efficient tumor treatment effect on human breast cancer cells and tumor-bearing mouse models.

Description

Technical Field

[0001] The invention relates to the technical field of tumor treatment, in particular to a copper silicate-based nano targeted drug-carrying system for collaboratively 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, it still faces many challenges in practical application. Therefore, further improving the efficiency of the reaction to increase the generation 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 oxidative damage caused by excessive production of reactive oxygen species (ROS). In view of this, one strategy to kill cancer cells is usually to disrupt their redox homeostasis, on the one hand to produce higher levels of reactive oxygen species (ROS) and enhance the oxidative stress of cells, and on the other hand to weaken the antioxidant capacity of cells and make their defense function deficient. However, most current studies focus 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) to overcome cancer chemoresistance by disrupting cellular redox homeostasis. CuHPT oxidizes endogenous GSH in cells into oxidized glutathione (GSSG), effectively consuming GSH, while the decomposition of the CuHPT structure releases Cu. + and HPT, Cu + Catalyzes H2O2 to generate hydroxyl radicals (·OH), and HPT promotes O2 ·- The unique metal organic framework structure and ROS amplification effect of CuHPT effectively inhibit the growth of drug-resistant colorectal tumors and double the survival time of tumor mice.

[0004] Carbon quantum dots (CDs) refer to carbon nanoparticles with high dispersion and size less than 10nm. CDs have unique photoluminescence properties (related to size and excitation wavelength), low cytotoxicity and good thermal stability, so they have broad application prospects in research fields such as bioimaging, free radical scavenging, analytical sensing, disease diagnosis and treatment.

[0005] In recent years, biomass-derived CDs have received extensive attention due to their excellent properties, including low cost, abundant surface functional groups, environmental friendliness, biocompatibility, etc. Research in recent years has shown that biomass-derived CDs can mimic the structure and function of natural enzymes as nanozymes, especially glutathione oxidase, catalase, or superoxide dismutase. This unique enzyme-like property provides a new and effective approach for research and intervention related to oxidative stress in biological systems. Coffee-derived chlorogenic acid (ChA) CDs were prepared by a simple hydrothermal reaction, and they exhibited significant glutathione oxidase-like activity by reducing GSH to generate GSSG. Ferroptosis was induced in human liver tumor cells (HepG2) by promoting the unbalanced redox reaction caused by GSH depletion and inactivation of glutathione peroxidase 4 (GPX4).

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

[0007] The purpose of the present invention is to provide a copper silicate-based nano-targeted drug delivery system for the synergistic treatment of breast cancer, which is used for the synergistic treatment of breast cancer by disrupting the redox homeostasis. Due to the imbalance of intracellular redox homeostasis caused by oxidative stress and weakened antioxidant ability, the copper silicate-based nano-targeted drug delivery system shows high-efficiency tumor treatment effects in human breast cancer cells and tumor-bearing mouse models.

[0008] To achieve the above object, the present invention provides a copper silicate-based nano-targeted drug delivery system for the co-treatment of breast cancer, including 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 to generate CuSiO3@TAF@CDs, and then polyethylene glycol is covalently modified on the surface of CuSiO3@TAF@CDs to obtain CuSiO3@TAF@CDs-PEG, and hyaluronic acid with targeting ability is coated on the surface of CuSiO3@TAF@CDs-PEG to prepare the 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, centrifuge to collect the product Cu2O, wash with ethanol, and disperse in ethanol;

[0012] B. Synthesis of Cu2O@mSiO2: Dilute the above Cu2O dispersion in a mixture of ethanol and deionized water, successively add 25 wt% cetyltrimethylammonium chloride aqueous solution and triethylamine diluted 10 times with ethanol, add tetraethyl orthosilicate, stir the mixture at room temperature, collect the solid Cu2O@mSiO2 by centrifugation and washing, and redisperse in ethanol;

[0013] C. Synthesis of CuSiO3: Add 2-methylimidazole and ethanol to the Cu2O@mSiO2 dispersion, stir at room temperature, centrifuge and wash twice with ethanol to obtain CuSiO3, and redisperse the obtained CuSiO3 in ethanol for use.

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

[0015] Weigh the narcissus bulb powder and put it into a crucible, roast it in air at 200 °C for 2 h. After cooling, prepare it according to the mass ratio of carbonized narcissus powder to pure water of 1:100, then ultrasonicate at room temperature for 1 h. After the ultrasonication ends, centrifuge to collect the supernatant, and filter it with a microporous membrane with a pore size of 0.22 μm to obtain a brown clear solution. Then carry out dialysis purification, and finally put it into a freeze dryer for drying to obtain the narcissus-derived carbon quantum dot powder.

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

[0017] Add tamoxifen powder and an aqueous solution of narcissus-derived carbon quantum dots to the CuSiO3 ethanol dispersion according to the mass ratio of CuSiO3: tamoxifen: narcissus-derived carbon quantum dots of 2:1:1, ultrasonically disperse evenly, stir overnight at room temperature, centrifuge to collect the precipitate, and thus obtain CuSiO3@TAF@CDs.

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

[0019] Disperse CuSiO3@TAF@CDs in ethanol, add silane polyethylene glycol according to the mass ratio of CuSiO3: polyethylene glycol of 1:5, ultrasonically disperse evenly, react at 60 °C for 24 h, centrifuge to collect the precipitate, and thus obtain CuSiO3@TAF@CDs-PEG.

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

[0021] Disperse CuSiO3@TAF@CDs-PEG in ethanol, add an aqueous solution of hyaluronic acid according to the mass ratio of CuSiO3: hyaluronic acid of 2:1, stir overnight at room temperature, centrifuge to collect the precipitate, and thus obtain CuSiO3@TAF@CDs-PEG-HA.

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

[0023] 1. The present invention develops a CuSiO3@TAF@CDs-PEG-HA (CTCPH) nano-targeted drug delivery system for the synergistic treatment of breast cancer by disrupting the redox homeostasis. By loading the drug TAF on CuSiO3, the drug loading rate is 30.8%, the intracellular pH is reduced by upregulating the lactic acid content, 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 and HA is introduced as a tumor targeting agent. The targeted CTCPH enters human breast cancer cells and accumulatively releases 83.7% of TAF and 49.3% of Cu within 24 h of acidic degradation 2+ , and the Cu ion-mediated Fenton-like reaction promotes ROS generation. The TAF-mediated reduction of intracellular pH can enhance ROS generation. In addition, the released CDs act as GSH-oxidizing nanoenzymes to promote the consumption of the antioxidant GSH.

[0024] 2. Based on the imbalance of intracellular redox homeostasis caused by oxidative stress and weakened antioxidant capacity, CTCPH exhibits highly efficient tumor treatment effects in both human breast cancer cells and tumor-bearing mouse models, with a tumor inhibition rate of 76.4%. The H&E staining results of major organs also indicate that CTCPH has reliable biosafety and is expected to become an innovative strategy for precision tumor treatment.

[0025] The technical solutions of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Brief Description of the Drawings

[0026] Figure 1 It is the synthesis and characterization of CDs of the present invention, where (a) is the transmission electron microscopy image of CDs, (b) is the particle size distribution diagram of CDs, (c) is the X-ray diffraction spectrum of CDs, (d) is the AFM image and thickness distribution of CDs, and (e) is the three-dimensional AFM image of CDs;

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

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

[0029] Figure 4 It is the UV-vis spectrum and corresponding standard curve diagram of different concentrations of TAF of the present invention. Among them, (a) is the UV-vis spectrum of different concentrations of TAF, and (b) is the standard curve corresponding to TAF;

[0030] Figure 5 It is the test result diagram of in vitro acid-responsive release of the present invention. Among them, (a) is the TEM image of CTCPH after soaking at pH = 5.4 for 24 h, (b) is the locally enlarged image of (a), (c) is the cumulative release curve of TAF from CTCPH at different pH values, and (d) is the cumulative release curve of Cu ions from CTCPH at different pH values;

[0031] Figure 6 It is the test result diagram of GSH consumption and ·OH of the present invention. Among them, (a) is the mechanism diagram of the GSH oxidase-like activity of CDs, (b) is the change result diagram of the absorbance with time when CDs and GSH are added, (c) is the Michaelis-Menten equation curve, and (d) is the double-reciprocal plot;

[0032] Figure 7are the results of the consumption of GSH by CTCPH of the present invention and the generation of ·OH by CTCPH under different conditions, where (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 the mechanism diagram of the consumption of GSH and the generation of ROS by CTCPH, (d) is the degradation of MB after treatment with CTCPH under different pH conditions, (e) is the degradation of MB after treatment with CTCPH for different times under the condition of pH = 5.4, and (f) is the ESR spectrum corresponding to different pH conditions under CTCPH treatment;

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

[0034] Figure 9 are the result diagrams of testing the cytotoxicity of CTCPH by the CCK-8 assay of the present invention. Among them, (a) is the evaluation of the activity of MCF-7 cells, (b) is the evaluation of the activity 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 the CLSM image of MCF-7 cells after incubation with CTCPH for 6 h of the present invention;

[0036] Figure 11 are the results of the evaluation of the in vivo antitumor effect and biosafety of the present invention. Among them, (a) is the establishment and treatment plan of the MCF-7 tumor model, (b) are the representative photos of tumors in different groups after 14 days of treatment, (c) is the tumor volume change curve of different groups during treatment, and (d) is the body weight change curve of mice in different groups during treatment;

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

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

[0039] Unless otherwise defined, the technical terms or scientific terms used in the present invention shall have the ordinary meanings understood by those of ordinary skill in the field to which the present invention pertains.

[0040] Unless otherwise defined, the reagents, equipment and other materials used in the present invention are all obtained from regular commercial sources.

[0041] Example 1

[0042] A copper silicate-based nano-targeted drug delivery system for the co-treatment of breast cancer, comprising 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 by electrostatic interaction to generate CuSiO3@TAF@CDs, and then polyethylene glycol (PEG) is covalently modified on the surface of CuSiO3@TAF@CDs to obtain CuSiO3@TAF@CDs-PEG (CTCP), and hyaluronic acid (HA) with targeting ability is coated on the surface of CuSiO3@TAF@CDs-PEG (CTCP) to prepare the CuSiO3@TAF@CDs-PEG-HA (CTCPH) nano-targeted drug delivery system.

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

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

[0045] A. Synthesis of Cu2O: Dissolve 0.1 mmol of Cu(NO3)2 and 0.12 mmol of NH4NO3 in 40 mL of deionized water. Then, add 2.0 mL of NaOH (0.2 M) solution at a specific rate of 1.8 mL / min. After continuing to stir for 10 min, add 3.5 mL of ascorbic acid (0.1 M) solution at a specific rate of 0.6 mL / min, and stir for another 10 min. After the reaction is completed, collect the product Cu2O by centrifugation, wash it with ethanol, and disperse it in 10 mL of ethanol. -1 B. Synthesis of Cu2O@mSiO2: Dilute the above Cu2O dispersion in a mixture of 20 mL of ethanol and 10 mL of deionized water. Subsequently, add 0.16 mL of 25 wt% cetyltrimethylammonium chloride (CTAC) aqueous solution and 0.15 mL of triethylamine (TEA) diluted 10 times with ethanol in sequence. After adding 50 μL of tetraethyl orthosilicate (TEOS), stir the mixture at room temperature for 14 h. Collect the solid Cu2O@mSiO2 by centrifugation and washing, and redisperse it in 10 mL of ethanol.

[0046]

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

[0048] CuSiO3 is a layered CuSiO3 nanoflower carrier.

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

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

[0051] Weigh the narcissus bulb powder and put it into a crucible. Bake it in air at 200 °C for 2 h. After cooling, prepare it according to the mass ratio of carbonized narcissus powder to pure water of 1:100. Then, ultrasonicate it at room temperature for 1 h. After the ultrasonication, centrifuge to collect the supernatant, and filter it with a microporous filter membrane with a pore size of 0.22 μm to obtain a brown clear solution. Then, perform dialysis purification, and finally put it into a freeze dryer to dry, obtaining narcissus-derived carbon quantum dots (CDs) powder.

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

[0053] Add tamoxifen powder and an aqueous solution of narcissus-derived carbon quantum dots to the CuSiO3 ethanol dispersion according to the mass ratio of CuSiO3:tamoxifen:narcissus-derived carbon quantum dots of 2:1:1. Ultrasonically disperse it evenly, stir overnight at room temperature, and centrifuge to collect the precipitate, namely CuSiO3@TAF@CDs.

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

[0055] Disperse CuSiO3@TAF@CDs in ethanol, add silane polyethylene glycol according to the mass ratio of CuSiO3: polyethylene glycol of 1:5, ultrasonically disperse it evenly, react at 60 °C for 24 h, and centrifuge to collect the precipitate, namely CuSiO3@TAF@CDs-PEG.

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

[0057] Disperse CuSiO3@TAF@CDs-PEG in ethanol, add an aqueous solution of hyaluronic acid according to the mass ratio of CuSiO3 to hyaluronic acid of 2:1, stir overnight at room temperature, and centrifuge to collect the precipitate to obtain CuSiO3@TAF@CDs-PEG-HA.

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

[0059] Example 2

[0060] Verify the performance of the synthesized CuSiO3@TAF@CDs-PEG-HA.

[0061] 1. Synthesis and characterization of CTCPH.

[0062] CDs were obtained after a series of treatments using daffodil bulbs as the precursor. First, the morphology and size distribution of the CDs particles were studied by transmission electron microscopy (TEM). The CDs are uniformly dispersed spherical nanoparticles without obvious aggregation ( Figure 1 in (a)). High-resolution shows that these CDs have obvious lattice fringes with a lattice fringe spacing of 0.21 nm, corresponding to the (100) crystal plane of graphite carbon. According to the particle size distribution statistics, the average particle size of the CDs is 2.4 ± 0.3 nm ( Figure 1 in (b)). To further clarify its structural characteristics, X-ray diffraction (XRD) was used to analyze the crystal configuration of the CDs ( Figure 1 in (c)). It can be found that a broad diffraction peak appears at 20° for the CDs, which can be attributed to the diffraction plane of amorphous graphite carbon, indicating the successful preparation of the CDs. In addition, the surface morphology of the 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 ultraviolet-visible (UV-vis) spectrum of the CDs is as Figure 2As shown in (a). CDs exhibit a broad ultraviolet-visible absorption between 200 and 550 nm, with strong light absorption ability; 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 orbitals of the carbon ring and is related to the (crystalline) carbon core. As Figure 2 shown in (b), CDs exhibit excitation-dependent fluorescence (photoluminescence, PL) in the range of 320 - 400 nm, with the optimal excitation wavelength being 364 nm and the optimal emission wavelength being 439 nm. The inset of the figure shows the photos of the CDs aqueous solution and the pure aqueous solution under 365 nm ultraviolet radiation. It can be seen that compared with pure water, the CDs aqueous solution produces significant blue fluorescence under ultraviolet excitation. The above results indicate that CDs have good fluorescence properties.

[0064] After fixing TAF and CDs nanoparticles on CuSiO3 through electrostatic interaction, PEG was modified by covalent interaction and further coated with HA to prepare the CuSiO3@TAF@CDs-PEG-HA (CTCPH) nano-targeted drug delivery system. A large number of uniformly dispersed CDs nanoparticles can be seen in the TEM image, and CuSiO3 still retains the complete morphology of the nanoflower without being damaged by the loading and functionalization steps ( Figure 3 (a)). The XRD pattern of CTCPH ( Figure 3 (b)) did not change significantly compared with CuSiO3, indicating that the basic crystal phase structure of CuSiO3 was not affected by the loading and functionalization modification. The Fourier transform infrared (FTIR) spectrum ( Figure 3 (c)) shows that a characteristic peak belonging to C═O appears at 1040 cm -1 , confirming the successful coating of HA. In addition, an absorption peak at 3132 cm -1 appears in the spectrum, which is considered to be the stretching vibration of -CH2- in PEG, further proving the successful polyethylene glycolylation of CuSiO3. In addition, the change in Zeta potential further proves the successful preparation of the CTCPH nanocomposite system ( Figure 3 (d)).

[0065] 2. The drug loading capacity of CTCPH.

[0066] Figure 4The absorption curves of TAF solutions with different concentrations in the UV-Vis spectrum are shown. These curves correspond to the absorption of TAF solutions with different concentrations (4 - 60 μg / mL) in the wavelength range of 200 - 400 nm. The ultraviolet characteristic peak of TAF is at 238 nm. The standard curve of the figure shows the linear relationship between the TAF concentration and the 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 2 value of the linear regression is 0.999, indicating a high linear correlation between the concentration and the absorption value. Through the TAF standard curve, the concentration of TAF can be inferred from the absorption value of the supernatant of the drug-loaded nanomaterial in subsequent experiments.

[0067] The drug loading rate and the loading efficiency are two key indicators to describe the degree of drug loading in the drug delivery system. 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 the loading efficiency are as high as possible. Table 1 shows the TAF drug loading rate and loading efficiency when using the CuSiO3 carrier to load TAF. It includes five different initial concentrations of TAF (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 with the decrease of the initial concentration. Considering comprehensively, the TAF loading concentration of 1 mg / mL was selected for further analysis and application in subsequent experiments.

[0068] Table 1 Results of CuSiO3 loaded with TAF

[0069] <![CDATA[Initial concentration of TAF (mg / mL -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-responsive release test.

[0071] As Figure 5 shown, similar to CuSiO3, CTCPH also exhibits acid-responsive degradation behavior. Under weak acidic conditions (pH = 5.4), after incubation for 24 h, the nanoflower structure of CTCPH dissociates and decomposes significantly into fragments. The results further confirm that this nanodrug 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 respectively. As Figure 5 shown, CTCPH exhibits excellent chemical stability under normal physiological conditions, and only 14.7% of TAF and 7.4% of Cu ions are released within 24 h respectively. In contrast, CTCPH shows an obvious drug burst release pattern under simulated tumor weak acidic conditions, and the cumulative release of TAF and Cu within 24 h 2+They were 83.7% and 49.3% respectively. Therefore, it can be speculated that CTCPH maintains its structural integrity during neutral blood circulation and rapidly releases TAF and Cu ions in situ after entering weakly acidic tumor tissues, thus effectively reducing the non-specific release of TAF and Cu.

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

[0073] Figure 6 Figure (a) shows a schematic diagram of the GSH oxidase-like activity of CDs. Reduced glutathione (GSH) is converted to oxidized glutathione (GSSG) through the catalysis of CDs, and at the same time, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) reacts to form a yellow product containing nitrite (TNB). Figure 6 Figure (b) shows the change in absorbance at 412 nm with time at different concentrations of GSH (from 0.5 to 10 mM) in a system containing CDs (100 μg / mL) and DTNB (100 μM). It can be seen from the figure that as the concentration of GSH increases, the absorbance also increases, and as the time extends, the absorbance decreases, indicating that GSH is effectively scavenged by CDs. Subsequently, quantitative analysis of enzyme kinetics showed that CDs exhibited typical Michaelis-Menten kinetics characteristics in the DTNB color reaction ( Figure 6 Figure (c)). In addition, the V of CDs was determined by double-reciprocal plot analysis max and K m were 4.38×10-6 M and 2.72×10-7 M·min respectively -1 ( Figure 6 Figure (d)). These results indicate that CDs exhibit GSH oxidase-like activity under experimental conditions, and its kinetic characteristics conform to the classical Michaelis-Menten model.

[0074] Incubate different concentrations of CTCPH (50, 100, and 150 μg / mL) with an excess of GSH, and then use DTNB-containing to measure the remaining GSH. As the concentration increases, the specific absorbance at 412 nm decreases, indicating that CTCPH can effectively consume GSH ( Figure 7 Figure (a)), which is attributed to the large amount of Cu 2+ released that can oxidize GSH to GSSG. At the same time. In addition, compared with CTPH without loaded CDs (CuSiO3@TAF-PEG-HA), CTCPH shows enhanced GSH consumption ability ( Figure 7 Figure (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), with the decrease of pH value, the absorbance of MB gradually decreased, indicating that MB was gradually degraded and the amount of ·OH generated increased. Figure 7 (e) shows the change in the absorbance of MB at different time points (1, 6, 12, and 24 h) 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 CTCPH under different pH conditions are shown in Figure 2. 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, which can be attributed to the increase in environmental acidity, which also promotes the degradation of Cu in CTCPH. 2+ Mediated Fenton-like reaction rate.

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

[0077] Tamoxifen (TAF), as an anti-estrogen drug, is 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 the level of intracellular ROS. First, the activity of 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 by the 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 with a characteristic absorption peak at 570nm. Figure 8As shown in (b), compared with the untreated control cell group, the lactic acid content in CTCPH-treated cells increased relatively, rising to about 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 of human breast cancer cell line (MCF-7) cells ( Figure 8 in (c)). BCECF-AM is highly sensitive to pH, and its green fluorescence intensity decreases with increasing acidity. It can be seen from the confocal laser scanning microscope (CLSM) images that the green fluorescent signal of MCF-7 cells in the CTCPH group was significantly weaker than that of the blank control group and the CCPH group without loaded TAF, demonstrating that CTCPH did induce an enhancement of intracellular acidity. The ROS generation performance of MCF-7 cells under different treatment conditions was further evaluated using the fluorescent probe (DCFH-DA). Figure 8 As can be seen from (d), the ROS green fluorescent signal of the control group was almost negligible, indicating a relatively low ROS level, while significant fluorescent signals were observed in cells treated with CCPH and CTCPH. Especially in the CTCPH group, the ROS fluorescent signal was the strongest, showing an enhanced ROS generation ability. The above studies have shown that the inhibition of complex I activity induced by TAF loaded in CTCPH can specifically reduce the intracellular pH, thus overcoming the problem of inherent acidity deficiency in tumors, improving the efficiency of Cu ion-mediated Fenton-like reaction, and enhancing the ROS level.

[0079] 6. Cytotoxicity detection and cell uptake.

[0080] Given the enhanced GSH consumption and increased ROS generation induced by CTCPH, the cytotoxicity of CTCPH was further tested by the CCK-8 assay ( Figure 9(a)). TAF, CuSiO3, CTCP (CuSiO3@TAF@CDs-PEG), and CTCPH were co-cultured with CTCPH cells for 24 h, respectively, to investigate their cytotoxic effects on MCF-7 cells. The results showed that all treatments exhibited dose-dependent inhibitory effects on MCF-7. The individual TAF and CuSiO3 carriers showed certain cytotoxicity, which was related to TAF-mediated chemotherapy and chemodynamic therapy mediated by the released Cu ions. However, the CTCP group and the CTCPH group showed higher cytotoxicity, indicating that the loaded CDs and TAF jointly exerted a synergistic effect with the CuSiO3 carrier. Especially in the CTCPH group, as the concentration of CTCPH increased to 100 μg / mL, the cell viability of MCF-7 cells was lower than 20%, showing the lowest cell viability 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 enhanced the cytotoxicity to MCF-7 cells. In addition, the cytotoxicity of CTCPH to normal cells (HUVEC) was not obvious ( Figure 9 (b)). At a treatment concentration of 100 μg / mL, the cell viability remained above 90%. This was because of the specific degradation and release behavior of CTCPH in the tumor microenvironment, resulting in less release of Cu ions and TAF in normal cells, thus generating less ROS compared to MCF-7. The above results indicate that CTCPH has a good selective therapeutic effect on MCF-7 cells.

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

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

[0083] The mechanism of action of CTCPH can be summarized as follows: (1) After being taken up by MCF-7 cells through targeted action, the acidic microenvironment promotes the degradation of CTCPH, resulting in the release of TAF, CDs, and Cu ions from CTCPH; (2) The released Cu 2+React with high levels of intracellular GSH to generate Cu + and GSSG, resulting in a significant depletion of the antioxidant GSH, while Cu + undergoes a Fenton-like reaction with H2O2 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 increase lactate levels, leading to increased acidification within tumor cells and accelerating the Fenton-like reaction between Cu + and H2O2, generating more ·OH within tumor cells and enhancing the oxidative stress of tumor cells. Through the above mechanisms of action, CTCPH can exert the synergistic effects of its components, disrupting the intracellular redox homeostasis and thus achieving the killing of cancer cells.

[0084] 8. Evaluation of anti-tumor effects and biosafety in vivo.

[0085] To systematically evaluate the therapeutic effect of CTCPH, an MCF-7 nude mouse tumor model was first established, and the following substances were injected via the tail vein respectively: phosphate buffered saline (PBS), TAF, CuSiO3, CTCP, and CTCPH ( Figure 11 as shown in (a) below). 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. The pictures of the solid tumors of each group after 14 days of treatment are shown in Figure 11 as shown in (b) below. Compared with the PBS control group, the CuSiO3 or TAF treatment groups had a partial inhibitory effect on tumor growth. Notably, compared with the groups injected with CuSiO3 or TAF, the tumor-bearing mice treated with CTCPH achieved the best therapeutic effect, which was due to the synergistic therapeutic effect and HA targeting caused by the redox homeostasis imbalance resulting from enhanced ROS and GSH depletion. From the tumor growth curve ( Figure 11 as shown in (c) below), it can be seen that compared with the PBS control group with rapid tumor growth, free TAF had limited drug effects due to low tumor accumulation, while the CTCP group or CTCPH group showed significant tumor growth inhibition effects. In particular, due to the HA targeting effect, injection of CTCPH showed a stronger growth inhibitory effect on tumors, with a tumor inhibition rate of 76.4%, which further confirmed that CTCPH has excellent anti-tumor effects.

[0086] During the entire treatment period, the body weights of all groups of MCF-7 tumor-bearing mice increased to a certain extent ( Figure 11In (d) of the figure, this indicates that all treatments and dosages have little adverse effect on the normal growth of mice. Subsequently, H&E staining and TUNEL staining were performed on the tumor tissues collected from different treatment groups, and it was confirmed that CTCPH has significant tumor treatment effects ( Figure 12 ). As expected, in the H&E and TUNEL staining experiments, the CTCPH group showed the most severe histological damage among all groups. In addition, no obvious pathological abnormalities were observed in the main organs stained with H&E in each group, which indicates that the injected CTCPH has little toxic and 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 biochemistry and complete blood cell analysis were respectively performed on the blood samples collected from normal nude mice after injecting CTCPH and PBS to detect a series of key parameters. As shown in Table 2, compared with the control group, there were no significant changes in the main indicators of blood biochemistry and blood routine in the mice receiving CTCPH. The above results indicate 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 (μmol / L) 15.20±1.05 13.88±1.25 10.91～85.09

[0089] In the present invention, first, narcissus bulbs were used as precursors, and carbon dots (CDs) were obtained by high-temperature heat treatment. It was observed by TEM that the CDs were uniformly dispersed spherical nanoparticles with an average particle size of 2.4 ± 0.3 nm and obvious lattice fringes. The fluorescence spectrum indicated that the CDs had good fluorescence properties. Through electrostatic interaction and covalent modification, TAF, CDs, PEG, and HA were respectively loaded and modified on the surface of CuSiO3, and the CuSiO3@TAF@CDs-PEG-HA (CTCPH) nano-targeted drug delivery system was successfully prepared. Analyses such as TEM, XRD, and FTIR confirmed the successful preparation and functional modification of CTCPH. Through UV-vis spectroscopy and the standard curve, the loading capacity of CTCPH for the drug TAF was determined, and finally, a TAF concentration of 1 mg mL -1 was selected for subsequent experiments. The drug loading rate was 30.8%, and the drug loading efficiency was 92.5%. The in vitro acid-responsive release test showed that CTCPH exhibited an obvious drug burst release pattern under weak acidic conditions (pH = 5.4), and 83.7% of TAF and 49.3% of Cu 2+ were cumulatively released within 24 h, indicating its pH-dependent degradation characteristics. In addition, the GSH oxidase-like activity of CDs was verified, which could effectively consume GSH. And it was detected that CTCPH had enhanced GSH consumption ability and enhanced ·OH generation ability under weak acidic conditions.

[0090] Cell experiments showed that CTCPH enhanced 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 increasing the intracellular lactate content (increased to 1.8 times that of the control group). Cytotoxicity assays showed that CTCPH exhibited significant cytotoxicity against MCF-7 cells (cell viability was lower than 20% at 100 μg / mL -1 ), while showing lower toxicity against normal cells (HUVECs) (cell viability remained above 90%). Cell uptake experiments showed that CTCPH could be effectively taken up by MCF-7 cells. The mechanism of CTCPH-mediated cooperative cell death is mainly as follows: CTCPH degrades in MCF-7 to release TAF, CDs, and Cu 2+ , and Cu 2+ consumes GSH and is reduced to Cu + , and Cu + catalyzes the generation of ·OH from H2O2; in addition to its endocrine therapy effect, TAF can also reduce the intracellular pH, further promoting the generation of ·OH; CDs act as GSH oxidase to enhance GSH consumption, and the enhanced ROS and reduced GSH enhance intracellular oxidative stress, thus achieving a cooperative killing effect on MCF-7 cells. In vivo experiments showed that in the MCF-7 nude mouse tumor model, CTCPH exhibited significant tumor growth inhibition, with a tumor inhibition rate of 76.4%, and had low toxicity and side effects, showing reliable biosafety. Blood biochemistry and complete blood cell analysis further confirmed the biosafety of CTCPH, indicating its potential as a candidate drug for precision cancer therapy.

[0091] Therefore, the present invention uses the above-mentioned copper silicate-based nano-targeted drug delivery system for the cooperative treatment of breast cancer to disrupt the intracellular redox homeostasis. Due to the imbalance of intracellular redox homeostasis caused by oxidative stress and weakened antioxidant capacity, the copper silicate-based nano-targeted drug delivery system exhibits efficient tumor treatment effects in 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 and are not intended to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that they can still modify or equivalently replace the technical solutions of the present invention, and these modifications or equivalent replacements cannot make the modified technical solutions 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 the co-treatment of breast cancer, characterized in that: It includes 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 to generate CuSiO3@TAF@CDs, and then polyethylene glycol is covalently modified on the surface of CuSiO3@TAF@CDs to obtain CuSiO3@TAF@CDs-PEG, and hyaluronic acid with targeting ability is coated on the surface of CuSiO3@TAF@CDs-PEG to prepare the CuSiO3@TAF@CDs-PEG-HA nano-targeted drug delivery system.

2. The copper silicate-based nano-targeted drug delivery system for the co-treatment of breast cancer according to claim 1, wherein: The loading concentration of tamoxifen is 1 mg / mL.

3. The copper silicate-based nano-targeted drug delivery system for the co-treatment of breast cancer according to claim 1, wherein, The preparation method of the said 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, centrifuge to collect the product Cu2O, wash it with ethanol, and disperse it in ethanol; B. Synthesis of Cu2O@mSiO2: Dilute the above Cu2O dispersion in a mixed solution of ethanol and deionized water, successively add 25 wt% cetyltrimethylammonium chloride aqueous solution and triethylamine diluted 10 times with ethanol, add tetraethyl orthosilicate, stir the mixture at room temperature, collect the solid Cu2O@mSiO2 by centrifugation and washing, and redisperse it in ethanol; C. Synthesis of CuSiO3: Add 2-methylimidazole and ethanol to the Cu2O@mSiO2 dispersion, stir at room temperature, centrifuge and wash twice with ethanol to obtain CuSiO3, and redisperse the obtained CuSiO3 in ethanol for use.

4. The copper silicate-based nano-targeted drug delivery system for the collaborative treatment of breast cancer according to claim 1, wherein The preparation method of the narcissus-derived carbon quantum dots is as follows: Weigh the narcissus bulb powder and put it into a crucible, roast it in air at 200 °C for 2 h. After cooling, prepare it according to the mass ratio of carbonized narcissus powder to pure water of 1:100, then ultrasonicate it at room temperature for 1 h. After the ultrasonication ends, centrifuge to collect the supernatant, and filter it with a microporous filter membrane with a pore size of 0.22 μm to obtain a brown clear solution. Then carry out dialysis purification, and finally put it into a freeze dryer for drying to obtain the narcissus-derived carbon quantum dot powder.

5. The copper silicate-based nano-targeted drug delivery system for the co-treatment of breast cancer according to claim 1, wherein The preparation method of CuSiO3@TAF@CDs is as follows: Add tamoxifen powder and an aqueous solution of narcissus-derived carbon quantum dots to the CuSiO3 ethanol dispersion according to the mass ratio of CuSiO3:tamoxifen:narcissus-derived carbon quantum dots of 2:1:1, ultrasonically disperse it evenly, stir overnight at room temperature, centrifuge to collect the precipitate, and thus obtain CuSiO3@TAF@CDs.

6. The copper silicate-based nano-targeted drug delivery system for the co-treatment of breast cancer according to claim 1, characterized in that, The preparation method of CuSiO3@TAF@CDs-PEG is as follows: Disperse CuSiO3@TAF@CDs in ethanol, add silane polyethylene glycol according to the mass ratio of CuSiO3:polyethylene glycol of 1:5, ultrasonically disperse it evenly, react at 60 °C for 24 h, centrifuge to collect the precipitate, and thus obtain CuSiO3@TAF@CDs-PEG.

7. The copper silicate-based nano-targeted drug delivery system for the synergistic treatment of breast cancer according to claim 1, wherein The preparation method of CuSiO3@TAF@CDs-PEG-HA is as follows: Disperse CuSiO3@TAF@CDs-PEG in ethanol, add an aqueous solution of hyaluronic acid according to the mass ratio of CuSiO3 to hyaluronic acid of 2:1, stir overnight at room temperature, centrifuge to collect the precipitate, and thus obtain CuSiO3@TAF@CDs-PEG-HA.

Images