Carbon sphere confined copper-based nanoszyme, and preparation method and application thereof

Abstract

This invention provides a carbon-sphere-confined copper-based nanozyme, its preparation method, and its applications. The nanozyme comprises hollow carbon nanospheres, an ultrasmall copper sulfide nanozyme, glucose oxidase, and a nucleic acid aptamer. The hollow carbon nanospheres serve as the confinement carrier. The ultrasmall copper sulfide nanozyme and glucose oxidase are loaded onto the surface of the hollow carbon nanospheres. The nucleic acid aptamer is coupled to the surface of the polyethylene glycol-modified hollow carbon nanospheres loaded with the ultrasmall copper sulfide nanozyme and glucose oxidase. This nanozyme achieves a synergistic therapeutic mechanism of active targeting, self-supplied H2O2 cascade catalysis, GSH consumption, and photothermal enhancement, significantly inhibiting tumor cell proliferation and achieving tumor ablation in mice. It exhibits good biosafety and promising clinical application prospects.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a carbon sphere-confined copper-based nanozyme, its preparation method, and its application. Background Technology

[0002] Cancer is a major disease that seriously threatens human health, with its incidence and mortality rates continuing to rise. Traditional cancer treatments such as chemotherapy and radiotherapy suffer from significant toxic side effects, lack of specificity, and easy development of drug resistance. Catalytic therapy based on nanomaterials, especially nanozyme-mediated reactive oxygen species (ROS) catalytic therapy, has attracted much attention due to its high selectivity and low side effects. However, single nanozyme catalytic therapy faces several challenges: First, the level of endogenous hydrogen peroxide (H2O2) in the tumor microenvironment (TME) is insufficient, making it difficult to continuously and efficiently generate highly cytotoxic hydroxyl radicals (·OH); second, overexpressed glutathione (GSH) in the TME effectively scavenge the generated ROS, severely weakening the therapeutic effect. Therefore, developing intelligent responsive nanozyme systems that can self-supply H2O2 and simultaneously consume GSH is of great significance.

[0003] Currently, researchers are attempting to introduce glucose oxidase (GOx) to catalyze the oxidation of glucose within tumors to produce H2O2 and gluconic acid, thus addressing both the H2O2 deficiency and creating an acidic environment. Meanwhile, copper-based nanomaterials (such as CuS) possess mixed valence states (Cu... + / Cu 2+ It can not only catalyze the formation of ·OH from H2O2 through a Fenton-like reaction, but also utilize Cu 2+ GSH is consumed. However, the catalytic activity of CuS nanozymes is highly dependent on their particle size: the smaller the particle size, the higher the peroxidase-like activity, but ultrasmall nanoparticles are prone to severe aggregation due to their high surface energy, leading to a decrease in catalytic performance. In addition, although the photothermal effect can accelerate the catalytic reaction, the existing system still struggles to effectively integrate H2O2 self-supply, GSH consumption, controllable stabilization of nanozyme size, and photothermal enhancement effect simultaneously, and lacks active targeting capability, limiting its in vivo delivery efficiency and therapeutic efficacy.

[0004] Therefore, there is an urgent need to provide a carbon sphere-confined copper-based nanozyme, its preparation method, and its application to solve the problems of insufficient H2O2, excessive GSH consumption leading to ROS removal, easy aggregation and inactivation of nanozymes, and lack of active targeting in traditional nanozyme catalytic therapy. Summary of the Invention

[0005] This invention provides a carbon sphere-confined copper-based nanozyme, its preparation method, and its application. This nanozyme utilizes the confinement effect of hollow carbon spheres to stabilize ultra-small CuS nanoparticles, achieves a cascade catalytic reaction with self-supplied H2O2 through GOx loading, and significantly enhances the catalytic therapeutic effect of tumors by combining the active targeting of nucleic acid aptamers and photothermal enhancement.

[0006] This invention provides a carbon-confined copper-based nanozyme, comprising hollow carbon nanospheres (HCNs), ultrasmall copper sulfide (CuS) nanozymes, glucose oxidase (GOx), and a nucleic acid aptamer (Apt-M). The hollow carbon nanospheres serve as the confinement carrier. The ultrasmall copper sulfide nanozyme is loaded onto the surface of the hollow carbon nanospheres via in-situ reduction growth. The glucose oxidase is loaded onto the surface of the hollow carbon nanospheres via electrostatic adsorption. The nucleic acid aptamer, as a targeting molecule, is covalently coupled to the surface of the polyethylene glycol (PEG) modified hollow carbon nanospheres loaded with ultrasmall copper sulfide nanozymes and glucose oxidase via an amidation reaction.

[0007] According to the present invention, a copper-based nanozyme confined by carbon spheres is provided, wherein the particle size of the hollow carbon nanosphere material is 150-200 nm.

[0008] According to the present invention, a copper-based nanozyme confined to carbon spheres is provided, wherein the ultrasmall copper sulfide nanozyme is loaded onto the surface of hollow carbon nanospheres by in-situ reduction growth, and the loading amount of the ultrasmall copper sulfide nanozyme is 30 wt.% to 50 wt.% of the mass of the hollow carbon nanospheres.

[0009] According to the present invention, a copper-based nanozyme confined in carbon spheres is provided, wherein the glucose oxidase is loaded onto the surface of hollow carbon nanospheres through electrostatic adsorption, and the loading mass percentage of glucose oxidase is 15% to 30%.

[0010] According to the present invention, a carbon-sphere-confined copper-based nanozyme is provided, wherein the nucleic acid aptamer is covalently coupled to the surface of a polyethylene glycol-modified ultrasmall copper sulfide nanozyme via an amidation reaction, and the nucleic acid aptamer can specifically recognize the MUC1 protein, the sequence of which is: NH2-(CH2)6-GCAGTTGATCCTTTGGATACCCTGGTTTTTTTTTT.

[0011] The hollow carbon spheres (HCNs) possess a hollow spherical structure and a large specific surface area, which not only provides abundant loading sites as a nanocarrier, but more importantly, can effectively suppress the aggregation of ultra-small CuS nanoparticles through spatial confinement effects, maintaining their high catalytic activity. Simultaneously, the hollow carbon spheres themselves exhibit good biocompatibility and photothermal conversion performance.

[0012] The ultrasmall CuS nanozyme has a mixed valence state (Cu + / Cu 2+ Under the weakly acidic conditions of the tumor microenvironment, it can degrade and release Cu. + and Cu 2+ Among them, Cu + It can catalyze H2O2 to undergo a Fenton-like reaction to generate highly cytotoxic ·OH, achieving CDT; Cu 2+ This can consume the overexpressed GSH in tumor cells, reduce ROS clearance capacity, and further enhance oxidative stress damage.

[0013] The GOx can catalyze the oxidation of glucose in tumor cells to produce gluconic acid and H2O2. On the one hand, it enables the self-supply of H2O2, overcoming the limitation of insufficient H2O2 in the tumor microenvironment; on the other hand, the gluconic acid produced can lower the local pH value, providing a better acidic environment for Fenton-like reactions, thereby initiating cascade catalytic reactions.

[0014] The Apt-M can specifically recognize the MUC1 protein overexpressed on the surface of tumor cells, achieving active targeted delivery, improving the enrichment efficiency of nanozymes at the tumor site and prolonging their retention time, while reducing toxic side effects on normal tissues.

[0015] The principle of this invention's carbon-sphere-confined copper-based nanozyme for enhanced cascade catalytic therapy is as follows: Surface-modified nucleic acid aptamers specifically bind to tumor cells, achieving active targeted enrichment. Upon entering the tumor cells, the loaded GOx catalyzes the oxidation of glucose to produce H2O2 and gluconic acid. The ultrasmall CuS on the surface of the hollow carbon spheres degrades in an acidic microenvironment, releasing Cu... + The catalytic self-supplied H2O2 generates a large amount of ·OH for CDT therapy; simultaneously, Cu 2+ By consuming intracellular GSH and disrupting the redox balance, the killing effect of ROS is amplified. Under irradiation with an 808nm near-infrared laser, hollow carbon spheres and CuS synergistically generate excellent photothermal effects. On the one hand, they directly perform photothermal therapy (PTT); on the other hand, local heating accelerates the Fenton-like reaction rate, further enhancing the efficacy of CDT, forming a cascade amplified catalytic therapy of "self-supplied H2O2-GSH consumption-photothermal enhancement". In addition, the confinement effect of the hollow carbon spheres ensures the long-term stability and high catalytic activity of the ultrasmall CuS nanoparticles. Through active targeting and cascade catalysis, this nanozyme can significantly inhibit tumor cell proliferation, achieving efficient and safe tumor ablation.

[0016] A method for preparing copper-based nanozymes confined by carbon spheres as described above is provided, comprising the following steps: Step 1, Preparation of hollow carbon nanospheres: Hollow carbon nanospheres were synthesized using the hard template method; Step 2, In-situ loading of ultra-small copper sulfide nanozymes: The hollow carbon nanospheres obtained in Step 1 are dispersed in an aqueous solution containing CuCl2·2H2O. After stirring, NaS·9H2O is added, and an in-situ reduction growth reaction is carried out under stirring conditions to obtain hollow carbon nanospheres@CuS composite, i.e. HCNs@CuS composite (abbreviated as HC). Step 3, loading of glucose oxidase: The HCNs@CuS complex obtained in step 2 is dispersed in water, glucose oxidase is added, and loading is carried out by electrostatic adsorption to obtain the HCNs@CuS / GOx complex (abbreviated as HCG). Step 4, Surface polyethylene glycol modification: The HCNs@CuS / GOx composite obtained in step 3 is mixed and reacted with dicarboxylated polyethylene glycol to obtain a polyethylene glycol-modified composite. Step 5, coupling of nucleic acid aptamers: In the presence of EDC and NHS, the carboxyl groups on the surface of the complex obtained in step 4 are activated, and the nucleic acid aptamers are covalently coupled to the surface of the complex through an amidation reaction to obtain carbon sphere-confined copper-based nanozymes.

[0017] According to the present invention, a method for preparing a carbon-confined copper-based nanozyme is provided. The specific process for synthesizing hollow carbon nanospheres by hard template method in step 1 is as follows: ethanol, ammonia and deionized water are mixed, tetrapropoxysilane is used as the silicon source, and resorcinol and formaldehyde are used as the carbon source. After obtaining the precursor, it is carbonized at 700°C for 3 hours in a nitrogen atmosphere, and the silicon dioxide template is removed by etching with hydrofluoric acid for 4 hours.

[0018] According to the method for preparing copper-based nanozymes confined within carbon spheres provided by the present invention, the conditions for the in-situ reduction growth reaction in step 2 are: a reaction temperature of 90°C and a reaction time of 30 min. These conditions facilitate the uniform and dense in-situ growth of CuS nanoparticles on the surface of hollow carbon spheres, forming nanozymes with ultra-small particle size and uniform distribution.

[0019] According to the method for preparing a carbon sphere-confined copper-based nanozyme provided by the present invention, the loading percentage of glucose oxidase in step 3 is 15% to 30%, which is determined using a BCA protein quantification kit.

[0020] According to the method for preparing a carbon sphere-confined copper-based nanozyme provided by the present invention, the reaction time for PEGylation modification in step 4 is 24 h, and unbound dicarboxylic acid PEG is removed by centrifugation and washing.

[0021] According to the method for preparing a carbon sphere-confined copper-based nanozyme provided by the present invention, the specific steps of nucleic acid aptamer coupling in step 5 are as follows: EDC·HCl and NHS are added to HCGP dispersion, shaken and activated for 30 min, centrifuged and then the nucleic acid aptamer is added, the shaking reaction is continued for 5 h, and the target product HCGPM is obtained by centrifugation and washing.

[0022] This invention provides an application of the carbon sphere-confined copper-based nanozyme described above, specifically in the preparation of a drug for combined CDT and photothermal therapy of tumors. The combined therapy involves a cascade catalytic reaction initiated by glucose oxidase with self-supplied H2O2 to generate hydroxyl radicals, achieving CDT. Simultaneously, photothermal therapy is achieved through the photothermal effect of hollow carbon nanospheres and ultrasmall copper sulfide under near-infrared laser irradiation. Furthermore, Cu... 2+ The nanozyme enhances therapeutic efficacy by consuming GSH within tumor cells. Under near-infrared irradiation (808nm), the nanozyme achieves CDT-PTT cascade synergistic therapy through active targeting-mediated tumor enrichment, GOx self-supplied H2O2-induced cascade catalysis, CuS-mediated GSH consumption, and photothermally enhanced ·OH generation. This significantly inhibits tumor cell proliferation, achieves tumor ablation in mice, and demonstrates good therapeutic efficacy and biosafety, showing promising clinical application prospects in the field of tumor catalytic therapy.

[0023] The present invention provides a carbon sphere-confined copper-based nanozyme, its preparation method, and its application, which have the following beneficial effects: (1) By utilizing the spatial confinement effect of hollow carbon spheres, ultra-small CuS nanoparticles were stably anchored on the surface of carbon spheres through in-situ reduction growth, effectively solving the key technical problem of easy aggregation and inactivation of ultra-small particle size nanozymes, while maintaining their high peroxidase-like catalytic activity. This nanozyme constructed a self-supplied H2O2 cascade catalytic system by electrostatically adsorbing and loading GOx, which can convert endogenous glucose in tumors into H2O2 and further generate highly cytotoxic ·OH, overcoming the limitation of insufficient H2O2 in the tumor microenvironment on catalytic therapy. Surface modification with Apt-M enabled active targeted delivery to MUC1-overexpressing tumor cells, significantly improving the accumulation and retention efficiency of nanozymes at the tumor site and reducing the toxic side effects of non-specific distribution on normal tissues.

[0024] (2) Utilizing the mixed valence state characteristics of CuS (Cu + / Cu 2+This targeted nanozyme system, while achieving Fenton-like catalysis to generate ·OH, effectively consumes GSH overexpressed in tumor cells, disrupting intracellular redox balance and further amplifying the oxidative damage effect mediated by reactive oxygen species. Under 808 nm near-infrared laser irradiation, hollow carbon spheres and CuS synergistically exhibit excellent photothermal conversion performance. On the one hand, it directly achieves PTT (photothermal-to-thermal conversion), and on the other hand, the local heating significantly accelerates the Fenton-like reaction rate and enhances the CDT (photothermal-to-thermal conversion) effect, forming a triple cascade amplified catalytic therapy mode of "self-supplied H2O2 - GSH consumption - photothermal enhancement". This targeted nanozyme system fully utilizes the synergistic combination of the confinement and photothermal properties of hollow carbon spheres, the Fenton catalysis and GSH consumption capabilities of CuS, the H2O2 self-supplied performance of GOx, and the active targeting capability of nucleic acid aptamers. It has excellent tumor targeting, high catalytic activity, and good biosafety, enabling precise, efficient, and low-toxicity treatment of tumors.

[0025] (3) The application of the carbon-sphere-confined copper-based nanozyme provided by this invention is a non-invasive, highly efficient, and low-toxicity tumor catalytic therapy method. When using this drug, the localized heating generated by laser irradiation can not only directly kill tumor cells but also significantly increase the efficiency of Cu... + The GOx-mediated Fenton-like reaction efficiency enhances CDT efficacy; simultaneously, GOx-mediated H2O2 self-supply provides ample substrates for CDT, while GSH consumption blocks the antioxidant defense mechanisms of tumor cells. This cascade catalytic therapy modality is not only significantly stronger than single catalytic therapy or photothermal therapy, but also superior to simply combining the two. More importantly, it avoids the multidrug resistance and systemic toxicity associated with traditional chemotherapy, exhibiting extremely high tumor selectivity and promising clinical application prospects. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0027] Figure 1 This is a schematic diagram illustrating the preparation process of a carbon sphere-confined copper-based nanozyme, its preparation method, and its application in breast cancer treatment. Figure 2 This is a TEM image of HC in Example 1 of the present invention, which describes a carbon sphere-confined copper-based nanozyme, its preparation method, and its application (illustration: HRTEM image of HC). Figure 3 This is an EDS diagram of HC in Example 1 of the present invention, which describes a carbon sphere-confined copper-based nanozyme, its preparation method, and its application. Figure 4 This is the XPS analysis diagram of HC in Example 1 of the present invention, which describes a carbon sphere-confined copper-based nanozyme, its preparation method, and its application. Figure 5 The infrared spectra of H, HC, HCG, HCGP, and HCGPM in Example 1 of the present invention, which describes a carbon sphere-confined copper-based nanozyme, its preparation method, and its application. Figure 6 This is a Zeta potential diagram of H, HC, HCG, HCGP, and HCGPM in Example 1 of the present invention, which describes a carbon sphere-confined copper-based nanozyme, its preparation method, and its application. Figure 7 This is a hydration kinetics diagram of H, HC, HCG, HCGP, and HCGPM in Example 1 of the present invention, which describes a carbon sphere-confined copper-based nanozyme, its preparation method, and its application. Figure 8 This invention relates to a carbon sphere-confined copper-based nanozyme, its preparation method, and the DNS UV-Vis absorption spectrum after HCGPM catalysis in Example 2. Figure 9 This is a graph showing the pH change of glucose catalyzed by HCGPM in Example 2 of the present invention, which describes a carbon sphere-confined copper-based nanozyme, its preparation method, and its application. Figure 10 This invention relates to a carbon-sphere-confined copper-based nanozyme, its preparation method, and application example 2, showing the generation of H2O2 under different concentrations of HCGPM. Figure 11 This invention relates to a carbon sphere-confined copper-based nanozyme, its preparation method, and application example 2, showing the UV-Vis absorption spectra of TMB catalyzed by HCGPM in the presence of different substrates. Figure 12 This invention relates to a carbon sphere-confined copper-based nanozyme, its preparation method, and application examples 2, showing the electron spin resonance (ESR) results of ·OH generated by different nanomaterials. Figure 13 This is a graph showing the glutathione-consuming capacity of different concentrations of HCGPM in Example 2 of the present invention, which describes a carbon-sphere-confined copper-based nanozyme, its preparation method, and its application. Figure 14 This is a graph showing the ability of HCGPM to catalyze the degradation of MB in a carbon sphere-confined copper-based nanozyme, its preparation method, and application example 2 of this invention. Figure 15 This invention relates to a carbon sphere-confined copper-based nanozyme, its preparation method, and application example 2, showing the heating and cooling curves of HCGPM at 808 nm wavelength and its photothermal conversion efficiency. Figure 16This invention relates to a carbon sphere-confined copper-based nanozyme, its preparation method, and the UV-Vis absorption spectra of ·OH generated under different laser power densities in Example 2. Figure 17 This is a diagram of HCGPM uptake by cells in Example 3 of the present invention, which describes a carbon sphere-confined copper-based nanozyme, its preparation method, and its application. Figure 18 This invention relates to a carbon-sphere-confined copper-based nanozyme, its preparation method, and its application. Example 3 shows MTT plots of MCF-7 cells treated under different conditions. Figure 19 This invention relates to a carbon sphere-confined copper-based nanozyme, its preparation method, and its application. Example 3 shows MCF-7 cells treated under different conditions and stained with live / dead cells (CLSM images). Figure 20 This is a graph showing the temperature change of mouse tumor tissue under different conditions after laser irradiation in Example 4 of the present invention, which describes a carbon sphere confined copper-based nanozyme, its preparation method, and its application. Figure 21 This is a graph showing the weight changes of tumor-bearing mice in different groups after treatment under different conditions in Example 4 of the present invention, which describes a carbon sphere-confined copper-based nanozyme, its preparation method, and its application. Figure 22 This is a graph showing the changes in average tumor volume of tumor-bearing mice under different conditions after treatment with a carbon sphere-confined copper-based nanozyme, its preparation method, and application example 4 of this invention. Figure 23 This invention relates to a carbon sphere-confined copper-based nanozyme, its preparation method, and application examples. 4 shows tumor images of tumor-bearing mice treated under different conditions. Figure 24 This invention relates to a carbon sphere-confined copper-based nanozyme, its preparation method, and application example 4. Histological evaluation of tumor tissues excised from tumor-bearing mice under different conditions after treatment, including H&E staining, TUNEL staining, and Ki-67 staining. Figure 25 These are hematoxylin-eosin stained tissue sections of the main organs (heart, liver, spleen, lungs, and kidneys) of tumor-bearing mice after different treatments in Example 4 of the present invention, which describes a carbon sphere-confined copper-based nanozyme, its preparation method, and its application. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. The components of the embodiments of the present invention described and shown in the accompanying drawings can generally be arranged and designed in various different configurations.

[0029] Therefore, the following detailed description of the embodiments of the invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the invention without inventive effort are within the scope of protection of the invention.

[0030] It should be noted that similar reference numerals and letters in the following figures indicate similar items; therefore, once an item is defined in one figure, it does not need to be further defined and explained in subsequent figures. Furthermore, the terms "first," "second," etc., are used only to distinguish descriptions and should not be construed as indicating or implying relative importance.

[0031] In the description of the embodiments of the present invention, it should be noted that the terms "inner", "outer", "upper", etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, or the orientation or positional relationship in which the product of the invention is usually placed when in use. They are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limiting the present invention.

[0032] Example 1

[0033] A carbon-confined copper-based nanozyme (HCGPM) uses hollow carbon nanospheres (HCNs, or H for short) as a carrier. Ultra-small CuS nanoparticles are loaded onto the surface of the hollow carbon nanospheres through in-situ reduction growth. GOx is further loaded through electrostatic adsorption, and finally, MUC-1Apt-M is covalently coupled through an amidation reaction to enable it to have active targeting capabilities.

[0034] The preparation method of this carbon sphere-confined copper-based nanozyme includes the following steps: Step 1, Preparation of HCNs: HCNs were synthesized using a hard template method. First, ethanol (17.5 mL), NH3·H2O (0.75 mL, 25 wt.%), and deionized water (2.50 mL) were mixed and stirred at 30 °C for 5 min. Then, tetrapropoxysilane (TPOS, 0.91 mL) was added and stirred for 15 min. Next, resorcinol (0.10 g) and formaldehyde (0.14 mL, 37 wt.%) were added and stirred for 24 h. The resulting mixture was centrifuged, and the precipitate was washed and dried in air. The resulting product was then carbonized at 700 °C for 3 h under a nitrogen atmosphere. Finally, the silica template was completely removed by HF etching for 4 h to obtain the HCNs.

[0035] Step 2, In-situ loading of ultrasmall CuS nanozymes: The HCNs (10 mg) obtained in Step 1 were dispersed in 20 mL of deionized water containing CuCl2·2H2O (7.7 mg) and stirred for 5 min. Then, NaS·9H2O (10.8 mg) was added, and the resulting suspension was stirred at 90 °C for 30 min to carry out an in-situ reduction growth reaction. After the reaction was completed, the precipitate was collected by centrifugation and washed three times with deionized water to obtain the HCNs@CuS complex (abbreviated as HC).

[0036] Step 3, GOx loading: HC (10 mg) obtained in Step 2 was dispersed in 10 mL of deionized water, and GOx (5 mg) was added. The mixture was shaken and reacted at 4 °C for 12 h. GOx was loaded onto the HC surface via electrostatic adsorption. After the reaction, the precipitate was collected by centrifugation and washed three times with deionized water to obtain the HCNs@CuS / GOx complex (HCG). The GOx loading was determined using a BCA protein quantification kit, and the GOx loading percentage was 23 wt.%.

[0037] Step 4, PEG surface modification: The HCG (3.0 mg) obtained in Step 3 was ultrasonically dispersed in 10.0 mL of distilled water, and COOH-PEG-COOH (6.0 mg) was added. The mixture was stirred and reacted for 24 h. After the reaction was completed, the mixture was centrifuged and washed to remove unbound dicarboxylic acid PEG, yielding the PEG-modified HCGP complex (HCGP for short).

[0038] Step 5, conjugation of nucleic acid aptamers: HCGP (1.0 mL, 2.0 mg / mL) obtained in step 4 was activated with EDC·HCl (500 µL, 500 mM) and NHS (500 µL, 100 mM). -1 The reaction mixture was magnetically stirred for 30 min, and the activated HCGP material was obtained by centrifugation and washed three times. Apt-M (10 OD) was denatured at 85 °C for 10 min, and then annealed in an ice bath for 10 min. The activated HCGP material was reacted with the annealed Apt-M under shaking conditions for 5 h, centrifuged, and washed three times with water to obtain the Apt-M-modified carbon sphere-confined copper-based nanozyme, named HCGPM. Simultaneously, for control experiments, a random sequence (Apt-C) without targeting properties was reacted under the same conditions to prepare HCGPC material.

[0039] Depend on Figure 2TEM images show that HCGPM consists of hollow spherical carbon shells with an average diameter of about 170 nm, with a large number of ultra-small CuS nanoparticles attached to the surface of the carbon spheres. High-resolution transmission electron microscopy (HRTEM) shows that the CuS nanoparticles have clear lattice fringes with a spacing of about 0.19 nm, corresponding to the (110) crystal plane of CuS. Figure 3 The elemental distribution map shows that C is highly concentrated in the shell region, while Cu and S are uniformly dispersed around the carbon signal, indicating that CuS was successfully loaded onto the carbon sphere surface. Figure 4 X-ray photoelectron spectroscopy (XPS) analysis showed two main peaks in the Cu2p spectrum at 933.5 eV and 953.3 eV, indicating that Cu is present in HC. + and Cu 2+ Mixed valence states. Fourier transform infrared spectroscopy (FT-IR) analysis, such as... Figure 5 As shown, HCG is at ~1070 cm⁻¹ -1 A new characteristic peak appeared at [value missing], attributed to the CH bending vibration of the benzene ring, confirming the successful loading of GOx. HCGPM was at ~1659 cm⁻¹. -1 The appearance of a new amide bond characteristic peak (C=O stretching vibration) indicates that Apt-M has been successfully covalently bonded to the HCGP surface. The Zeta potential test results are as follows... Figure 6 As shown, the Zeta potential of HCG was -35.33 mV, while that of HCGP decreased to -39.4 mV, and that of HCGPM decreased to -41.2 mV, indicating that COOH-PEG-COOH was successfully coated onto the HCG surface. Dynamic light scattering (DLS) analysis is as follows: Figure 7 As shown, the hydrated particle size of HC is approximately 187 nm, while the hydrated particle size of HCGPM increases to approximately 337 nm, further confirming the successful preparation of HCGPM.

[0040] Example 2

[0041] The ability of HCGPM to catalyze glucose was determined using the 3,5-dinitrosalicylic acid (DNS) colorimetric method to evaluate GOx activity. HCGPM (100 µg / mL) was used as the catalyst. -1 The sample was reacted with glucose (10 mM) in PBS buffer at pH 5.0, and the absorbance was measured after DNS color development. The results are as follows: Figure 8 As shown, the glucose concentration in the HCGPM group decreased significantly. Simultaneously, the pH of the reaction solution decreased from 7.4 to approximately 4.5. Figure 9 As shown, GOx successfully catalyzes the oxidation of glucose to gluconic acid.

[0042] The H2O2 generating capacity was evaluated using the Ti(SO4)2 colorimetric method. Different concentrations of HCGPM (0-200 µg / mL) were used.-1 The sample was reacted with glucose (10 mM) in PBS at pH 5.0, and the absorbance at 405 nm was measured after the addition of Ti(SO4)2. The results are as follows: Figure 10 As shown, the amount of H2O2 generated is dependent on the concentrations of HCGPM and glucose, confirming that HCGPM can sustainably supply H2O2.

[0043] The POD-like activity of HCGPM was evaluated using the TMB colorimetric method. The reaction system contained HCGPM (100 µg / mL). -1 The absorbance at 652 nm was measured after reacting TMB (1 mM) and H2O2 (2 mM) or glucose (10 mM) at pH 5.0. The results are as follows: Figure 11 As shown, the HCGPM+glucose group exhibits a significant ox-TMB absorption peak, indicating that the GOx-supplied H2O2 was successfully catalyzed by CuS to convert to ·OH. The ESR spectrum using DMPO as a trap is shown below. Figure 12 As shown, the HCGPM+glucose group exhibits a characteristic 1:2:2:1 quartet signal, clearly confirming the formation of ·OH.

[0044] The GSH-consuming capacity of HCGPM was evaluated using the DTNB method. Different concentrations of HCGPM (20-100 µg / mL) were tested. -1 The sample was reacted with GSH solution for 10 min, and then DTNB was added. The absorbance at 412 nm was then measured. The results are as follows: Figure 13 As shown, HCGPM consumes GSH in a concentration-dependent manner. Experiments using methylene blue (MB) as a probe, such as... Figure 14 As shown, the degradation rate of MB accelerated in the presence of 2.5–10.0 mM GSH, confirming that GSH consumption further enhanced ·OH generation.

[0045] Photothermal performance and photothermal enhanced catalysis evaluation: HCGPM dispersion (200 µg mL) was used. -1 Using an 808 nm laser (1.0 W cm⁻¹) -2 Irradiate for 10 minutes, and record temperature changes simultaneously. Results are as follows: Figure 15 As shown, the HCGPM dispersion exhibits excellent photothermal stability, with a calculated photothermal conversion efficiency (η) of 42.1%. The TMB colorimetric experiment is as follows... Figure 16 As shown, the absorbance of ox-TMB gradually increases with the increase of laser power density, confirming that the photothermal effect can significantly enhance the ·OH generation rate.

[0046] Example 3

[0047] Human breast cancer MCF-7 cells (MUC1 positive) and liver cancer HepG2 cells (MUC1 negative) were seeded into 6-well plates (1×10⁻⁶ cells per well). 5 Incubate in cells / well for 24 h. Replace the medium with Rh-B-labeled HCGPM (HCGPMR, 100 µg / mL). -1 Cells were incubated in fresh culture medium for 1, 2, 4, and 6 hours, respectively, and then washed with PBS. Cell uptake was analyzed by flow cytometry and confocal laser scanning microscopy (CLSM).

[0048] Depend on Figure 17 As can be seen from the results, the fluorescence signal of HCGPMR in MCF-7 cells gradually increased with the extension of incubation time, reaching a peak at 4h; while the fluorescence intensity in HepG2 cells was significantly lower than that in MCF-7 cells, indicating that Apt-M-mediated active targeting effectively enhanced the specific uptake of HCGPM in MUC1 positive cells.

[0049] Cytotoxicity was evaluated using the MTT assay. MCF-7 cells were seeded in 96-well plates and cultured for 24 h. Different nanomaterials (HC, HCG, HCGP, HCGPM, 100 µg / mL) were then added. -1 Incubation for 4 hours. The laser irradiation group used an 808nm laser (1.0 W cm⁻¹). -2 Irradiated for 5 min, and cultured for another 8 h before measuring cell viability. The experimental groups were as follows: (I) Control group (PBS), (II) Single catalytic therapy group (HCP), (III) Cascade catalytic therapy group (HCGP), (IV) Single-target enhanced cascade catalytic therapy group (HCGPM), (V) Single photothermal enhanced cascade catalytic therapy group (HCGP+L), and (VI) Enhanced cascade catalytic therapy group (HCGPM+L). Figure 18 As can be seen, the HCGPM+L group (VI) had the lowest cell viability, at only 23.0%, significantly lower than the HCGPM group (IV) and the HCGP+L group (V). Live / dead cell staining results are shown below. Figure 19 As shown, the HCGPM+L group exhibited strong red fluorescence (dead cells) and almost no green fluorescence (live cells), indicating that it had the most significant anti-tumor effect.

[0050] Example 4

[0051] A subcutaneous tumor model in nude mice was established using MCF-7 cells. 100 µL of MCF-7 cells (5 × 10⁻⁶ cells) were suspended in PBS. 7 Injected subcutaneously into the groin of mice (cells / mL). When the tumor volume reached approximately 100 mm... 3Mice were randomly divided into six groups (n=5) and injected with different nanomaterials via the tail vein: (I) PBS control group, (II) HCP group, (III) HCGP group, (IV) HCGPM group, (V) HCGP+L group, and (VI) HCGPM+L group. The dosage was 3 mg / kg. -1 (Calculated as CuS). Six hours after drug administration, the tumor sites of mice in groups (V) and (VI) were irradiated with an 808 nm laser (1.0 W cm⁻¹). -2 (5 min), while using an infrared thermal imager to record temperature changes.

[0052] like Figure 20 As shown, the tumor temperature in the HCGPM+L group increased to 56.1℃ within 10 min, which was significantly higher than that in the HCGP+L group (49.7℃), indicating that HCGPM still maintains good photothermal conversion performance in vivo.

[0053] Tumor volume and mouse weight were measured every two days during treatment. Figure 21 As shown, there was no significant difference in the body weight of mice in each group, indicating that the nanomaterials have good biocompatibility. Figure 22 The results showed that the HCGPM+L group (VI) exhibited the most significant tumor growth inhibition, with a tumor inhibition rate as high as 95.4%. After treatment (day 17), the mice were sacrificed and the tumor tissue was removed. Figure 23 The HCGPM+L group had the smallest tumor volume. Tumor tissue sections were stained with H&E (…). Figure 24 The results showed that the HCGPM+L group exhibited the most severe nuclear shrinkage and cell damage; TUNEL staining showed the strongest apoptosis signal in this group; Ki-67 staining showed the weakest cell proliferation signal in this group. H&E staining of major organs (heart, liver, spleen, lung, kidney) showed... Figure 25 As shown, no obvious abnormalities were observed, confirming the good biosafety of this nanozyme system.

[0054] In this embodiment, the sequences of the nucleic acid aptamers used are as follows:

[0055] Therefore, this invention provides a carbon-sphere-confined copper-based nanozyme, its preparation method, and its applications. Utilizing the spatial confinement effect of hollow carbon spheres, ultra-small CuS nanoparticles are stably anchored on the carbon sphere surface through in-situ reduction growth, effectively solving the key technical problem of easy aggregation and inactivation of ultra-small particle nanozymes, while maintaining their high peroxidase-like catalytic activity. This nanozyme, through electrostatic adsorption and loading of GOx, constructs a self-supplied H2O2 cascade catalytic system, capable of converting endogenous glucose in tumors into H2O2 and further generating highly cytotoxic ·OH, overcoming the limitations of insufficient H2O2 in the tumor microenvironment on catalytic therapy. Surface modification using Apt-M enables active targeted delivery to MUC1-overexpressing tumor cells, significantly improving the accumulation and retention efficiency of the nanozyme at the tumor site and reducing the toxic side effects of non-specific distribution on normal tissues.

[0056] Utilizing the mixed valence state characteristics of CuS (Cu + / Cu 2+ This targeted nanozyme system, while achieving Fenton-like catalysis to generate ·OH, effectively consumes GSH overexpressed in tumor cells, disrupting intracellular redox balance and further amplifying the oxidative damage effect mediated by reactive oxygen species. Under 808 nm near-infrared laser irradiation, hollow carbon spheres and CuS synergistically exhibit excellent photothermal conversion performance. On the one hand, it directly achieves PTT (photothermal-to-thermal conversion), and on the other hand, the local heating significantly accelerates the Fenton-like reaction rate and enhances the CDT (photothermal-to-thermal conversion) effect, forming a triple cascade amplified catalytic therapy mode of "self-supplied H2O2 - GSH consumption - photothermal enhancement". This targeted nanozyme system fully utilizes the synergistic combination of the confinement and photothermal properties of hollow carbon spheres, the Fenton catalysis and GSH consumption capabilities of CuS, the H2O2 self-supplied performance of GOx, and the active targeting capability of nucleic acid aptamers. It has excellent tumor targeting, high catalytic activity, and good biosafety, enabling precise, efficient, and low-toxicity treatment of tumors.

[0057] The application of a carbon-sphere-confined copper-based nanozyme provided by this invention is a non-invasive, highly efficient, and low-toxicity tumor catalytic therapy method. When using this drug, the localized heating generated by laser irradiation can not only directly kill tumor cells but also significantly enhance the Cu... + The GOx-mediated Fenton-like reaction efficiency enhances CDT efficacy; simultaneously, GOx-mediated H2O2 self-supply provides ample substrates for CDT, while GSH consumption blocks the antioxidant defense mechanisms of tumor cells. This cascade catalytic therapy modality is not only significantly stronger than single catalytic therapy or photothermal therapy, but also superior to simply combining the two. More importantly, it avoids the multidrug resistance and systemic toxicity associated with traditional chemotherapy, exhibiting extremely high tumor selectivity and promising clinical application prospects.

[0058] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A copper-based nanozyme confined within carbon spheres, characterized in that, The invention comprises hollow carbon nanospheres, ultrasmall copper sulfide nanozymes, glucose oxidase, and nucleic acid aptamers. The hollow carbon nanospheres serve as a confinement carrier. The ultrasmall copper sulfide nanozymes and glucose oxidase are loaded onto the surface of the hollow carbon nanospheres. The nucleic acid aptamers are coupled to the surface of the polyethylene glycol-modified hollow carbon nanospheres loaded with ultrasmall copper sulfide nanozymes and glucose oxidase.

2. The carbon sphere-confined copper-based nanozyme according to claim 1, characterized in that, The hollow carbon nanosphere material has a particle size of 150–200 nm.

3. The carbon sphere-confined copper-based nanozyme according to claim 1, characterized in that, The ultra-small copper sulfide nanozyme is loaded onto the surface of hollow carbon nanospheres via in-situ reduction growth, with the loading amount of the ultra-small copper sulfide nanozyme being 30 wt.% to 50 wt.% of the mass of the hollow carbon nanospheres.

4. The carbon sphere-confined copper-based nanozyme according to claim 1, characterized in that, The glucose oxidase is loaded onto the surface of hollow carbon nanospheres via electrostatic adsorption, with the glucose oxidase loading percentage ranging from 15% to 30%.

5. The carbon sphere-confined copper-based nanozyme according to claim 1, characterized in that, The nucleic acid aptamer is covalently coupled to the surface of a polyethylene glycol-modified ultrasmall copper sulfide nanozyme via an amidation reaction. The nucleic acid aptamer can specifically recognize the MUC1 protein, whose sequence is: NH2-(CH2)6-GCAGTTGATCCTTTGGATACCCTGGTTTTTTTTTT.

6. A method for preparing a copper-based nanozyme confined within carbon spheres as described in any one of claims 1-5, characterized in that, Includes the following steps: Step 1, Preparation of hollow carbon nanospheres: Hollow carbon nanospheres were synthesized using the hard template method; Step 2, In-situ loading of ultra-small copper sulfide nanozymes: The hollow carbon nanospheres obtained in Step 1 were dispersed in an aqueous solution containing CuCl2·2H2O, and NaS·9H2O was added after stirring. An in-situ reduction growth reaction was carried out under stirring conditions to obtain hollow carbon nanospheres@CuS composite. Step 3, loading of glucose oxidase: The HCNs@CuS complex obtained in step 2 is dispersed in water, glucose oxidase is added, and loading is carried out by electrostatic adsorption to obtain the HCNs@CuS / GOx complex. Step 4, Surface polyethylene glycol modification: The HCNs@CuS / GOx composite obtained in step 3 is mixed and reacted with dicarboxylated polyethylene glycol to obtain a polyethylene glycol-modified composite. Step 5, coupling of nucleic acid aptamers: In the presence of EDC and NHS, the carboxyl groups on the surface of the complex obtained in step 4 are activated, and the nucleic acid aptamers are covalently coupled to the surface of the complex through an amidation reaction to obtain carbon sphere-confined copper-based nanozymes.

7. The method for preparing carbon sphere-confined copper-based nanozymes according to claim 6, characterized in that, The specific process for synthesizing hollow carbon nanospheres using the hard template method in step 1 is as follows: ethanol, ammonia and deionized water are mixed, tetrapropoxysilane is used as the silicon source, and resorcinol and formaldehyde are used as the carbon source. After obtaining the precursor, it is carbonized at 700°C for 3 hours in a nitrogen atmosphere, and the silicon dioxide template is removed by etching with hydrofluoric acid for 4 hours.

8. The method for preparing carbon sphere-confined copper-based nanozymes according to claim 6, characterized in that, The conditions for the in-situ reduction growth reaction in step 2 are: a reaction temperature of 90°C and a reaction time of 30 min.

9. The method for preparing carbon sphere-confined copper-based nanozymes according to claim 6, characterized in that, In step 3, the glucose oxidase loading percentage is 15%–30%, and the BCA protein quantification kit is used for determination.

10. An application of a copper-based nanozyme confined by carbon spheres as described in any one of claims 1-5, characterized in that, It is used in the preparation of drugs for combined CDT and photothermal therapy of tumors.

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