Copper / temozolomide / metformin nano prodrug as well as preparation method and application thereof

Abstract

The invention relates to a copper / temozolomide / metformin nano prodrug as well as a preparation method and application thereof. The nano prodrug is formed by coordination self-assembly of temozolomide, copper ions and metformin, the mass fraction of temozolomide is 20%-60%, the mass fraction of copper ions is 20%-40%, and the mass fraction of metformin is 1%-60%. The invention provides a preparation method of a nano prodrug, which comprises the following steps: on the basis of a copper / temozolomide prodrug, introducing a metformin aqueous solution, and stirring and reacting in a water phase at normal temperature to obtain a three-component nano prodrug with glutathione response release characteristic. The prodrug is clear in structure and stable in particle size, copper ions and temozolomide can be released in a tumor microenvironment to induce copper death, and efficient treatment of drug-resistant glioblastoma is achieved in cooperation with metabolic intervention of metformin. Compared with the prior art, the preparation method disclosed by the invention is simple and convenient, an additional carrier is not needed, and the prodrug has good targeting property, biodegradability and treatment effect and has a good application prospect.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to an antitumor nanoprodrug constructed by metal ion coordination. Specifically, it relates to a copper / temozolomide / metformin nanoprodrug, its preparation method, and its application in the treatment of glioblastoma, belonging to the interdisciplinary technical fields of pharmaceutical formulation, prodrug technology, and tumor targeted therapy. Background Technology

[0002] Glioblastoma (GBM) is a high-grade malignant tumor that occurs in the central nervous system and is one of the most common and aggressive types of gliomas in clinical practice. This type of tumor is characterized by rapid growth, high recurrence rate, poor postoperative prognosis, and difficulty in treatment. Although current standard treatment strategies mainly include a combination of surgical resection, radiotherapy, and chemotherapy, the significant invasiveness of tumor cells often makes complete resection difficult, leading to treatment failure and a high recurrence rate.

[0003] Currently, temozolomide (TMZ) is the standard chemotherapy drug for treating glioblastoma. As a small molecule alkylating agent, TMZ can kill residual tumor tissue by inducing DNA strand breaks in tumor cells. However, the clinical response rate of glioblastoma patients to temozolomide is limited, with approximately 40% to 50% of patients exhibiting varying degrees of resistance during treatment, severely restricting its clinical efficacy and patient survival rates. Currently, most TMZ resistance mechanisms are related to the upregulation of DNA repair enzymes (such as MGMT) in tumor cells. Therefore, enhancing the sensitivity of glioblastoma to TMZ treatment and overcoming resistance are key technical challenges that urgently need to be addressed in this field.

[0004] In recent years, the emerging concept of copper death (Cuproptosis) has been proposed, offering a new therapeutic direction for overcoming drug resistance in gliomas. Copper death is a form of programmed cell death closely related to mitochondrial metabolism, primarily triggered by the abnormal accumulation of intracellular copper ions. Its mechanism involves the binding of copper ions to sulfur-containing acylated proteins (such as DLAT) in the TCA cycle, causing protein aggregation and loss of iron-sulfur cluster proteins, thereby inducing cellular stress responses and cell death. Previous studies have shown that copper ions can be used to induce copper death in certain drug-resistant tumor cells, thereby overcoming chemotherapy resistance limitations. However, the control of copper ion delivery, safety, and targeting remain major obstacles to its practical application.

[0005] Meanwhile, prodrug strategies have attracted widespread attention as an important means to improve the pharmacokinetic performance and bioavailability of small molecule drugs. Prodrugs are chemically modified or assembled to transform into active drugs in a specific in vivo environment, thereby achieving therapeutic advantages such as controlled release, targeted delivery, and low toxicity. However, most current prodrug carriers have complex structures, cumbersome preparation processes, and are prone to introducing additional nanocarrier systems. They also suffer from poor biocompatibility, difficult degradation, and weak targeting, limiting their widespread application in high-grade malignant tumors such as glioblastoma.

[0006] Existing research has made preliminary attempts to coordinate and assemble copper with certain drug molecules to construct carrier-free nanoprodrugs, achieving synergistic release of copper ions and drug components. However, problems remain, such as unstable particle size control, unclear structure, weak responsiveness, or a single therapeutic mechanism. In addition, there is a lack of systematic strategies to synergistically integrate copper death mechanisms with commonly used chemotherapy drugs temozolomide and metabolic regulators such as metformin, as well as a lack of precise intervention strategies for glioma drug resistance mechanisms.

[0007] Therefore, there is an urgent need to develop a novel copper-based nanoprodrug system with well-defined structure, stable particle size, good responsiveness, and multi-mechanism antitumor effects to achieve effective treatment of temozolomide-resistant gliomas, improve clinical treatment outcomes, and enhance patients' quality of life. Summary of the Invention

[0008] To overcome the problems of strong temozolomide resistance, significant toxic side effects, and poor drug targeting in the treatment of glioblastoma, this invention provides a novel copper / temozolomide / metformin nanoprodrug, its preparation method, and its application. This prodrug is constructed through the coordination of copper ions with drug molecules, eliminating the need for exogenous nanocarriers. It possesses advantages such as simple structure, mild reaction conditions, and good biodegradability. Furthermore, it exhibits glutathione-responsive release capability in the tumor microenvironment, synergistically inducing copper death and metabolic interference, thus achieving highly effective treatment for temozolomide-resistant gliomas. The following will specifically describe the prodrug structure, preparation process, and its application in tumor treatment, using different technical solutions of this invention.

[0009] In one embodiment of the present invention, a copper / temozolomide prodrug is provided, which is formed by the self-assembly of temozolomide and copper ions through coordination, wherein the mass fraction of temozolomide is 50%~90% and the mass fraction of copper ions is 10%~50%, and the prodrug has glutathione responsiveness.

[0010] Furthermore, the copper ions are divalent copper ions, which can form a stable chelate structure with the coordinating groups in temozolomide, thereby constructing a uniform nanoprodrug system.

[0011] In another embodiment of the present invention, a copper / temozolomide / metformin prodrug is provided, which is formed by coordination and assembly of temozolomide, copper ions and metformin, wherein: the mass fraction of temozolomide is 20%~60%, the mass fraction of copper ions is 20%~40%, and the mass fraction of metformin is 1%~60%, and the prodrug also has glutathione responsiveness.

[0012] Furthermore, the metformin is directly added to the copper / temozolomide prodrug solution, where a coordination reaction occurs with the participation of existing copper ions to form a three-component prodrug structure.

[0013] Preferably, both the copper / temozolomide prodrug and the copper / temozolomide / metformin prodrug have a nanosphere structure with a particle size range of approximately 5-32 nanometers, which is beneficial for the penetration and retention of tumor tissue (EPR effect).

[0014] In one embodiment of the present invention, a method for preparing a copper / temozolomide prodrug is provided, comprising the following steps: adding an aqueous solution of temozolomide to 10-120 mL of water, adding an aqueous solution of divalent copper salt, and then adding an aqueous solution of sodium hydroxide; stirring and reacting for 10 minutes to 24 hours at a reaction temperature of room temperature to 60°C; after the reaction is completed, removing the supernatant by centrifugation at 10000g for 10 minutes, resuspending the precipitate in water to obtain an aqueous prodrug solution, and drying to obtain a copper / temozolomide prodrug solid powder.

[0015] Further, the divalent copper salt can be any one of copper chloride, copper nitrate, or copper sulfate; the concentration of temozolomide is 1~5 mg / mL, the concentration of divalent copper salt is 50~800 mg / mL, the concentration of sodium hydroxide is 0.01~40 mg / mL, and the molar ratio of divalent copper salt to temozolomide is preferably 0.1~5:1.

[0016] In another embodiment of the present invention, a method for preparing a copper / temozolomide / metformin prodrug is provided, comprising the following steps: adding an aqueous solution of copper / temozolomide prodrug to 10-120 mL of water, adding an aqueous solution of metformin, stirring and reacting for 20 minutes to 24 hours at room temperature; then centrifuging to remove the supernatant, resuspending the precipitate in water to obtain an aqueous solution of the prodrug, and drying to obtain a solid powder of copper / temozolomide / metformin prodrug.

[0017] Optionally, the molar ratio of copper / temozolomide prodrug to metformin is 0.1 to 10:1, and the aqueous solution concentration of metformin is 0.1 to 50 mg / mL.

[0018] In one embodiment of the present invention, the copper / temozolomide prodrug or copper / temozolomide / metformin prodrug is used to prepare a drug for treating drug-resistant glioblastoma. The prodrug acts on glioma cells through a copper ion-induced copper death mechanism, thereby enhancing the killing ability against temozolomide-resistant cells.

[0019] Furthermore, the copper / temozolomide / metformin prodrug can respond to glutathione-triggered structural depolymerization in the tumor microenvironment, releasing the active drug temozolomide and copper ions, thereby achieving highly efficient targeted killing of tumor cells and synergistically improving the therapeutic effect.

[0020] Based on the above technical solution, the copper / temozolomide / metformin nanoprodrug of the present invention, its preparation method and application, involves reacting the clinically commonly used antitumor drug temozolomide (TMZ) with copper ions (Cu). 2+ Coordination to form copper / temozolomide nanoprodrugs, and further introduction of metformin with anti-metabolic regulation function, to construct a three-component self-assembled nanoprodrug system, effectively achieving targeted release and multi-mechanism synergistic therapy for glioblastoma (especially temozolomide-resistant glioma).

[0021] This nanoprodrug addresses key challenges in existing technologies through the following aspects: 1. Alleviating the problem of temozolomide resistance Glioblastoma is widely resistant to temozolomide chemotherapy, severely limiting its clinical efficacy. This invention utilizes the copper ion-induced "copper death" mechanism to significantly enhance the killing ability against drug-resistant glioma cells, overcoming the bottleneck of the single mechanism of action of traditional small molecule drugs.

[0022] 2. Utilizing nano-prodrugs to enhance targeting and controlled release performance The prodrug is constructed through the coordination interaction between copper ions and the drug, without the need for the introduction of a traditional carrier, forming a self-assembled structure with a nanoscale (5~30nm) and naturally possessing the EPR effect (enhanced permeation and retention effect), which can improve the accumulation of the drug at the tumor site; at the same time, the structure is responsive to glutathione (GSH), and can trigger structural dissociation in the high GSH environment of tumor cells, achieving precise controlled release.

[0023] 3. Synergistic multi-mechanism antitumor effect Based on copper death, this invention introduces metformin, which coordinates with copper to enhance the biological activity of copper ions and further interferes with the metabolic functions of cancer cells (such as the AMPK pathway). This enables multiple pathways to attack tumors, including metabolic reprogramming, apoptosis, and oxidative stress, thereby significantly improving the therapeutic effect.

[0024] 4. Green and simple synthesis strategy This invention uses an aqueous reaction system, and coordination self-assembly can be completed by stirring at room temperature to 60°C. The process does not require organic solvents or toxic reagents, and is simple to operate, mild, safe and environmentally friendly. The reaction yield is high, the process is controllable, and it has good potential for industrial scale-up.

[0025] 5. The prodrug structure is tunable and has strong responsiveness. The ratio of temozolomide, copper ions, and metformin can be adjusted under different molar ratios to regulate the structure and performance of the prodrug. In particular, it has a sensitive response to glutathione in the tumor microenvironment and can precisely release temozolomide and copper ions in cancer cells. The release process is time-dependent on the concentration, which helps to reduce toxicity to normal tissues and prolong the duration of action.

[0026] 6. Good in vitro cytotoxicity. According to CCK-8 cell activity assay, the prodrug prepared in this invention showed a significant concentration-dependent inhibitory effect in temozolomide-resistant glioblastoma cell lines (such as U87 / TR), which was significantly better than traditional TMZ monotherapy, showing good prospects for clinical translation.

[0027] In summary, the copper / temozolomide / metformin nanoprodrug and its preparation method provided by this invention not only achieve innovation in molecular design and synthesis pathways, but also demonstrate breakthrough advantages in tumor targeted therapy strategies. It has significant application value and industrialization potential, and is particularly suitable for the precision treatment of patients with drug-resistant glioblastoma, which is in line with the current development trend of precision medicine and targeted chemotherapy.

[0028] To more clearly illustrate the technical solution of the present invention, the embodiments of the present invention will be further described below with reference to the accompanying drawings. The accompanying drawings do not constitute a limitation of the present invention, but are intended to assist in understanding the present invention.

[0029] Figure 1 These are transmission electron microscopy (TEM) images of the copper / temozolomide prodrug, showing its nanoparticle size and quasi-spherical morphology.

[0030] Figure 2 The X-ray diffraction (XRD) patterns of the copper / temozolomide prodrug and the temozolomide monomer show that the prodrug has an amorphous structure.

[0031] Figure 3 These are the UV-Vis absorption spectra of copper / temozolomide prodrug, temozolomide, and copper nitrate solution, showing the changes in absorption peaks after coordination.

[0032] Figure 4 These are Fourier transform infrared (FTIR) spectra of copper / temozolomide prodrug and temozolomide, reflecting changes in functional groups and coordination interactions.

[0033] Figure 5 The image shows the X-ray photoelectron spectroscopy (XPS) spectrum of the copper / temozolomide prodrug, confirming that the copper element exists in the form of divalent copper.

[0034] Figure 6 These are transmission electron microscopy images of the copper / temozolomide / metformin prodrug, showing its particle size and morphological characteristics.

[0035] Figure 7 The images show the UV-Vis absorption spectra of copper / temozolomide prodrug, metformin, and their three-component prodrug, verifying that metformin participates in the assembly.

[0036] Figure 8 a is the UV absorption diagram of copper / temozolomide / metformin prodrug under different interaction disruptors, verifying its assembly driving mechanism; Figure 8 b is a particle size distribution diagram of dynamic light scattering (DLS) under the same conditions, illustrating the structural changes; Figure 8 c is a TEM image of the prodrug solution after processing, showing the particle state under the influence of coordination and non-covalent interactions.

[0037] Figure 9 The fluorescence spectrum of the copper / temozolomide / metformin prodrug reacted with glutathione and OPA, indicating its GSH responsiveness.

[0038] Figure 10 This is a TEM image of the prodrug in a glutathione solution, showing its depolymerization process under reducing conditions.

[0039] Figure 11 The image shows the UV-Vis absorption spectrum of the prodrug generating cuprous ions under glutathione conditions, confirming the release of cuprous ions.

[0040] Figure 12 a~12c illustrates the release behavior of temozolomide, including UV absorption spectra, standard curves, and release kinetic curves.

[0041] Figure 13 The image shows CCK-8 assay results of the cytotoxicity of copper / temozolomide prodrug and copper / temozolomide / metformin prodrug against U87 / TR cells, demonstrating their dose-dependent killing effects. Detailed Implementation

[0042] To better understand the technical solution and beneficial effects of the present invention, the prodrug structure design, preparation method, response characteristics, and biological performance of the present invention are described in detail below with reference to specific embodiments. It should be understood that the embodiments are only used to further illustrate the technical content of the present invention and do not constitute a limitation on the scope of protection of the present invention.

[0043] The specific embodiments of this invention mainly include two core contents: 1. Structural assembly, reaction parameter control, and morphological characterization of copper / temozolomide prodrug; 2. Metformin was introduced into the copper / temozolomide prodrug to construct a three-component copper / temozolomide / metformin prodrug, and its response release behavior and cytotoxic activity were systematically evaluated.

[0044] By comparing the preparation results under different reaction conditions and the functional differences with existing treatment strategies, the innovativeness and practical value of the nanoprodrug system of this invention in terms of structural controllability, biological responsiveness and synergistic therapy can be fully demonstrated.

[0045] In one possible implementation, the copper / temozolomide prodrug is formed by the self-assembly of temozolomide and copper ions through coordination, wherein the mass fraction of temozolomide is 50% to 90% and the mass fraction of copper ions is 10% to 50%, and the prodrug is glutathione responsive.

[0046] This implementation method achieves the construction of carrier-free nanoprodrugs by forming a stable chelate structure between the coordinating groups in temozolomide and copper ions. In an aqueous environment, temozolomide reacts with copper ions to form a coordination complex, which self-assembles into a nanoprodrug structure with a particle size of 5-30 nanometers. This structure has amorphous properties, which is beneficial to the stable existence of the drug in the physiological environment. This prodrug is responsive to glutathione. In the tumor microenvironment, the high concentration of reduced glutathione in cells can induce the depolymerization of this structure, releasing copper ions and temozolomide, thereby activating a dual mechanism of action of copper death and DNA damage, exerting a synergistic killing effect on malignant tumor cells such as glioblastoma.

[0047] Unlike traditional direct drug administration, this prodrug requires no additional nanocarrier, exhibiting higher biocompatibility and degradability. The introduction of copper ions not only enhances the antitumor efficacy of temozolomide but also significantly overcomes its resistance issues in glioma treatment. The prodrug's stable particle size control and excellent EPR (enhanced penetration and retention) effect facilitate targeted accumulation in tumor tissue. Furthermore, its glutathione responsiveness ensures precise release of the active ingredient within tumor tissue, enhancing therapeutic selectivity and reducing toxicity to normal tissues.

[0048] In this embodiment, different types of divalent copper salts can be selected as the copper ion source, including copper nitrate, copper chloride, or copper sulfate; the concentration of temozolomide aqueous solution can be adjusted within the range of 1-5 mg / mL to meet the needs of different formulations. The reaction temperature can be varied between room temperature and 60°C, and the stirring time can be adjusted from 10 minutes to 24 hours to obtain ideal particle size and structural stability. In addition, the amount of sodium hydroxide added can also regulate the pH environment for prodrug formation, thereby optimizing coordination efficiency. The prodrug structure can also achieve a balance between efficacy and toxicity by adjusting the copper ion ratio, adapting to different clinical treatment strategies.

[0049] Preferably, in the above embodiments, the copper ions in the copper / temozolomide prodrug are divalent copper ions.

[0050] This implementation method, based on the copper / temozolomide prodrug, explicitly specifies that the metal ion used is divalent copper ions (Cu). 2+ Divalent copper ions exhibit excellent coordination ability in aqueous solution, forming stable coordination complexes with nitrogen- or oxygen-containing coordinating groups (such as amides, ketones, or imine structures) in temozolomide molecules. This coordination drives self-assembly to form nanoparticle structures. Due to Cu... 2+ It possesses high charge density and a stable valence state, exhibiting good stability and bioresponsiveness during structure formation and subsequent drug release. In the presence of reduced glutathione, Cu... 2+ It can participate in redox reactions and enhance the responsiveness of prodrugs in the tumor microenvironment.

[0051] Using divalent copper ions as coordination centers helps improve the stability and uniformity of the prodrug assembly structure. Compared to other valence states of copper, such as monovalent copper (Cu), this method offers advantages over other valence states. + ) or metallic copper (Cu 0 The redox activity of divalent copper ions is more suitable for constructing prodrug structures with glutathione responsive properties. Furthermore, Cu... 2+ The metabolic and clearance pathways of drugs within organisms are relatively well-defined, resulting in higher safety and reducing the toxic side effects caused by non-specific releases, thereby enhancing the therapeutic window and clinical application value of drugs.

[0052] Different forms of divalent copper salts can be selected as the source of copper ions depending on the actual preparation conditions, including but not limited to copper nitrate (Cu(NO3)2), copper chloride (CuCl2), and copper sulfate (CuSO4). These copper salts can provide stable Cu ions in water. 2+ Ions. Cu under different pH conditions. 2+ The degree of coordination with temozolomide may differ, and appropriate pH adjustment can help optimize coordination efficiency. Although this embodiment preferably uses Cu... 2+It acts as a coordination center, but under certain specific conditions, it can also be transformed into Cu through a reduction control mechanism. + To enhance specific pharmacological effects, provided that the structural stability and biosafety of the prodrug are not affected.

[0053] In another embodiment, the copper / temozolomide / metformin prodrug is formed by coordination and assembly of temozolomide, copper ions, and metformin, wherein the mass fraction of temozolomide is 20% to 60%, the mass fraction of copper ions is 20% to 40%, and the mass fraction of metformin is 1% to 60%, and the prodrug is glutathione responsive.

[0054] In this embodiment, a copper / temozolomide / metformin prodrug forms a stable nanostructure through synergistic self-assembly of its three components. Specifically, temozolomide is first mixed with a divalent copper salt in an aqueous system, allowing temozolomide to coordinate with copper ions to form an initial copper-temozolomide prodrug solution. A metformin solution is then added to the copper-temozolomide prodrug solution system, further coordinating with copper ions within the system to construct a ternary nanoprodrug network structure with copper ions as the coordination center. This reaction occurs spontaneously at room temperature, ultimately forming stable nanoparticle structures with a particle size of approximately 30 nm. Because copper ions can simultaneously undergo multiple coordination interactions with both temozolomide and metformin, a dense and stable nanostructure is formed within the system, exhibiting a high responsiveness to the glutathione (GSH) environment. In the tumor microenvironment, increased endogenous GSH concentration disrupts the prodrug coordination structure, inducing the release of copper ions and temozolomide, while simultaneously activating the metabolic inhibitory effect of metformin, thereby achieving a synergistic therapeutic effect.

[0055] This three-component prodrug combines three synergistic anti-tumor mechanisms—copper death, DNA alkylation damage, and metabolic interference—significantly enhancing its killing effect on glioblastoma, particularly on temozolomide-resistant cells. The introduction of metformin not only enhances the assembly stability of the prodrug structure but also inhibits cancer cell metabolism through the AMPK pathway, improving therapeutic diversity and targeting. Furthermore, the coordination self-assembly process eliminates the need for exogenous nanocarriers, helping to reduce biotoxicity and preparation complexity, and improving biodegradability and in vivo safety. Glutathione responsiveness ensures that the drug is released primarily at the tumor site, reducing the risk of toxicity to normal tissues and improving therapeutic selectivity and clinical safety.

[0056] In this embodiment, copper ions can be provided by various divalent copper salts, such as copper nitrate, copper chloride, or copper sulfate. The metformin concentration can be adjusted within the range of 0.1–50 mg / mL according to the needs of the reaction system to achieve control over structural density and particle size. The mass fraction and molar ratio of temozolomide to copper and metformin can be adjusted according to pharmacodynamic requirements to achieve the emphasis of different therapeutic mechanisms. In addition, the reaction temperature (room temperature to 60°C) and stirring time (20 minutes to 24 hours) can be optimized according to actual needs to obtain a prodrug system with a more compact structure and more uniform particle size. At different concentrations, this three-component prodrug also exhibits significant dose-dependent cytotoxicity in vitro, which can be adapted to the treatment needs of different tumor drug resistance levels.

[0057] Furthermore, in this embodiment, metformin is directly added to the copper / temozolomide prodrug solution, where it undergoes a coordination reaction with the existing copper ions to construct a copper / temozolomide / metformin three-component prodrug structure. This method simplifies the operation process, avoids the pre-assembly step of intermediate complexes, and improves assembly efficiency and structural stability.

[0058] Under aqueous phase conditions, the guanidinium group in metformin molecules can react with Cu already present in the solution. 2+ Ions form a multidentate coordination structure, further stabilizing the three-dimensional configuration of the prodrug nanoparticles and improving structural density and particle size uniformity. Copper ions act as central coordination nodes in the prodrug structure, synergistically self-assembling the three components of temozolomide and metformin to form nanoscale prodrug particles. The resulting prodrug has stable particle size, good dispersibility, and strong biocompatibility and solution stability.

[0059] This three-component prodrug exhibits good glutathione (GSH) response characteristics in the tumor microenvironment. Under high GSH concentration conditions, it can undergo structural deconstruction to release three active ingredients: cuprous ions, temozolomide, and metformin. These ingredients induce copper death, DNA damage, and metabolic interference, respectively, thereby exerting a synergistic anti-tumor effect. It is especially suitable for glioblastoma cells with strong temozolomide resistance.

[0060] Common divalent copper salts, such as copper nitrate (Cu(NO3)2), copper chloride (CuCl2), or copper sulfate (CuSO4), can be used as the copper source. The molar ratio of metformin to copper ions can be adjusted within the range of 0.1 to 10:1 to control coordination efficiency and prodrug structural compactness. The reaction time can be adjusted from 20 minutes to 24 hours, and the reaction temperature can be set from room temperature to 60°C to further optimize the particle size control and structural maturity of the prodrug. This construction strategy has good versatility and scalability, which is conducive to the rapid development of nanoprodrug systems with controllable particle size, good responsiveness, and multi-mechanism synergistic therapeutic functions in different application scenarios. In a method for preparing a prodrug, the method includes the following steps: adding temozolomide aqueous solution to 10-120 mL of water, adding divalent copper salt aqueous solution, and then adding sodium hydroxide aqueous solution, stirring and reacting for 10 minutes to 24 hours, with the reaction temperature being room temperature to 60°C, centrifuging after the reaction, resuspending the precipitate to obtain copper / temozolomide prodrug aqueous solution, which can be further dried to obtain copper / temozolomide prodrug solid powder.

[0061] This implementation method constructs a copper / temozolomide prodrug nanostructure via an aqueous phase reaction. First, a temozolomide solution is added to deionized water to uniformly disperse the drug in the reaction system; then, a divalent copper salt solution is added dropwise to allow the Cu... 2+ A coordination reaction occurs with nitrogen- or oxygen-containing groups (such as amides or carbonyl groups) in the temozolomide molecule to form a preliminary coordination complex. The pH of the reaction solution is then adjusted by introducing sodium hydroxide, which helps improve the coordination ability and complexation stability of copper ions. The entire reaction process can be carried out at room temperature or under heating conditions, forming a stable nanostructured prodrug. After the reaction is complete, the product can be separated by centrifugation and then resuspended or dried to finally obtain a copper / temozolomide prodrug with a defined particle size and structure.

[0062] This preparation method is mild and environmentally friendly, employing an aqueous reaction system to avoid the use of organic solvents and high-temperature, high-pressure environments, thus exhibiting good process safety and environmental friendliness. Its reaction steps are simple, raw materials are readily available, and it facilitates large-scale production. Furthermore, by controlling the reaction time, temperature, and pH, the particle size and structural uniformity of the prodrug can be effectively controlled, improving the stability and therapeutic performance of the final product. This method does not rely on an external carrier, enabling the self-assembly of the drug and metal ions to form nanostructures, improving drug loading rate and controlled release, which helps to enhance efficacy and reduce toxic side effects.

[0063] The concentration of temozolomide can be adjusted within the range of 1–5 mg / mL to achieve different drug loading levels; different forms of divalent copper salt can be selected, such as copper nitrate, copper chloride, and copper sulfate; the concentration of sodium hydroxide can also be adjusted (0.01–40 mg / mL) for precise control of the system pH. The stirring time can be selected from 10 minutes to 24 hours according to reaction requirements, and the reaction temperature can be flexibly adjusted between room temperature and 60°C to meet the needs of different particle sizes and morphologies. The obtained nanostructures can be further freeze-dried or spray-dried to obtain solid powders for easy storage and subsequent formulation development.

[0064] Preferably, in the above preparation method, the divalent copper salt is one of copper chloride, copper nitrate or copper sulfate; the concentration of temozolomide is 1~5 mg / mL, the concentration of divalent copper salt is 50~800 mg / mL, the concentration of sodium hydroxide is 0.01~40 mg / mL, and the molar ratio of divalent copper salt to temozolomide is 0.1~5:1.

[0065] This embodiment, based on claim 5, further refines the types and proportions of key reaction components in the preparation of the copper / temozolomide prodrug. The selected divalent copper salt includes copper chloride, copper nitrate, or copper sulfate, which are readily soluble in water and provide stable Cu. 2+ Coordination center. By mixing temozolomide at concentrations of 1–5 mg / mL with an aqueous solution of copper salt at concentrations of 50–800 mg / mL under NaOH-controlled conditions (0.01–40 mg / mL), copper ions can effectively coordinate with the nitrogen-containing groups in temozolomide, ultimately constructing a nanoscale copper / temozolomide prodrug structure. The adjustable molar ratio (0.1–5:1) allows for significant regulation of the prodrug structure, particle size, and release properties, thus adapting to different drug release behaviors and therapeutic needs.

[0066] This embodiment enhances the controllability and reproducibility of the prodrug assembly reaction by limiting the concentration and ratio of raw materials. Within a specific molar ratio range, a prodrug structure with stable particle size and sufficient coordination can be achieved, effectively improving the loading efficiency and coordination stability of the prodrug. The copper salt used is widely available and inexpensive, suitable for industrial-scale production. Adjusting the pH by controlling the sodium hydroxide concentration further stabilizes the preparation system. Simultaneously, the tunable particle size and structure of the prodrug provides an important technological basis for subsequent applications such as targeted delivery, controlled-release regulation, and combination therapy.

[0067] Regarding the selection of divalent copper salts, in addition to copper chloride, copper nitrate, and copper sulfate, copper acetate and copper acetylacetonate, which have strong coordination ability and mild structures, can also be considered. In practice, the method of adding NaOH can be adjusted from single drop to slow drop to improve reaction uniformity. Adjusting the molar ratio range not only affects the prodrug structural density but also regulates the release rate of copper ions and the intensity of the "copper death" effect. For different indications or target requirements, structural modes with a high copper ratio (enhancing copper toxicity) or a high temozolomide ratio (enhancing DNA alkylation efficiency) can be preferred.

[0068] In another method for preparing the prodrug, the preparation method of the copper / temozolomide / metformin prodrug includes the following steps: adding the copper / temozolomide prodrug to water, adding metformin aqueous solution, stirring for 20 minutes to 24 hours, the reaction temperature is room temperature, centrifuging and resuspending the precipitate to obtain copper / temozolomide / metformin prodrug aqueous solution, which can be further dried to obtain copper / temozolomide / metformin prodrug solid powder.

[0069] This embodiment involves introducing metformin for secondary coordination assembly based on a copper / temozolomide prodrug. Specifically, the prepared copper / temozolomide nanoparticle prodrug solution is diluted or resuspended in an appropriate amount of deionized water, then metformin aqueous solution is added, and the mixture is stirred thoroughly at room temperature. The metformin molecule forms a new coordination bond with copper ions through its guanidine group, further coating or embedding the prodrug structure, ultimately forming a ternary assembled nanostructure with a particle size of approximately 30 nanometers. This reaction requires no heating or the use of organic solvents. After the reaction is complete, unreacted substances are removed by centrifugation, and the target product is obtained by resuspension or freeze-drying.

[0070] This preparation method is simple and operates under mild conditions, making it particularly suitable for drug systems containing temperature-sensitive components or requiring green formulation development. The three-component prodrug exhibits higher structural stability and greater biofunctional diversity, not only achieving synergistic tumor killing by copper ions and temozolomide but also introducing the interference of metformin on cancer cell metabolic pathways, significantly enhancing the overall therapeutic effect of the prodrug. Furthermore, the degree of assembly and particle size distribution can be influenced by adjusting the stirring time, facilitating fine structural control. The entire assembly process requires no high energy consumption or complex equipment, making it easy to promote and apply in laboratory or industrial environments.

[0071] Based on this process, the particle size and structural density of the final three-component prodrug can be controlled by adjusting the concentration of the copper / temozolomide prodrug. Metformin solution can be directly added to the copper / temozolomide prodrug solution to undergo a coordination reaction with existing copper ions, achieving efficient assembly of the three-component prodrug without the need for additional copper salts. The stirring time range (20 minutes to 24 hours) can be optimized according to the concentration and pH of the reaction system; under specific application requirements, the reaction can also be carried out under isothermal conditions (e.g., 30–40°C) to increase the rate. Furthermore, the resulting product can be used directly in aqueous solution or freeze-dried or spray-dried into a powdered prodrug for convenient storage, transportation, and further formulation development.

[0072] Furthermore, in this preparation method, the molar ratio of copper / temozolomide prodrug to metformin is 0.1~10:1, and the concentration of metformin is 0.1~50 mg / mL.

[0073] This implementation method, based on the preparation of copper / temozolomide prodrugs, further constructs a structurally homogeneous and functionally synergistic three-component prodrug system by adjusting the molar ratio of copper / temozolomide to metformin and the concentration of the latter. The molar ratio determines the proportion of metformin in the prodrug structure, directly affecting coordination equilibrium, prodrug particle size, encapsulation efficiency, and final release behavior. Controlling the metformin concentration within the range of 0.1–50 mg / mL helps achieve sufficient coordination, stable particle size, and controllable reaction during assembly. Within this parameter range, the reaction system exhibits good solubility and biocompatibility, spontaneously forming stable nanostructured prodrugs under room temperature aqueous conditions.

[0074] By adjusting the molar ratio and controlling the concentration, prodrug structures can be flexibly designed to adapt to different treatment strategies. For example, a low metformin ratio is suitable for a rapid killing mechanism focused on copper death and DNA alkylation, while a high ratio enhances its metabolic interference function, making it suitable for tumor cells with significant drug resistance. This approach enhances the structural regulation capability and functional adaptability of the prodrug system, improving targeted release efficiency and the precision of tumor treatment. Furthermore, an appropriate concentration range ensures controllable reaction efficiency and particle size, avoiding problems such as aggregation and crystallization, providing reliable parameters for industrial scale-up and consistency control.

[0075] Molar ratio adjustment is not limited to the copper / temozolomide / metformin ratio; it can be extended to other ratio adjustments in three-component systems, such as increasing the temozolomide ratio to enhance alkylation or increasing the copper ion ratio to enhance copper degradation. The metformin concentration can also be appropriately expanded or reduced based on solvent volume, prodrug loading, and pharmacodynamic requirements. To improve assembly efficiency, ultrasound, microwave, or heating can be used to promote coordination reactions, which is particularly suitable for industrial-scale preparation. Furthermore, without affecting activity, metformin can be replaced with structurally similar guanidine metabolism disruptors to achieve functional expansion.

[0076] In one embodiment, the copper / temozolomide prodrug or copper / temozolomide / metformin prodrug described above can be used to prepare a drug for treating drug-resistant glioblastoma, wherein the prodrug has a cytotoxic effect on temozolomide-resistant glioblastoma cells through a copper death mechanism.

[0077] This implementation applies the aforementioned nanoprodrug to the treatment of drug-resistant glioblastoma, achieving precise killing of cells unresponsive to traditional chemotherapy based on the "copper death" mechanism. After entering tumor cells, the copper / temozolomide prodrug releases active cuprous ions in a glutathione-reducing microenvironment. These cuprous ions accumulate intracellularly and specifically bind to lipoxygenated proteins in the mitochondrial TCA cycle, triggering protein aggregation, loss of iron-sulfur cluster proteins, and oxidative stress, ultimately inducing programmed cell death in tumor cells. This process is independent of the MGMT-mediated DNA repair pathway, thus also effective against temozolomide-resistant cells. The carrier-free nanostructure of this prodrug exhibits excellent EPR effects, enhancing its accumulation and retention in glioma tissue and improving therapeutic targeting.

[0078] Compared to temozolomide monotherapy, this prodrug effectively overcomes resistance mechanisms such as MGMT, significantly improving toxicity against drug-resistant glioma cells. Its copper death mechanism of action is independent of DNA damage responses, providing a powerful complement to temozolomide's mechanism of action and resulting in a superior treatment response for patients with drug-resistant glioblastoma. The prodrug's structure responds to the tumor microenvironment, releasing active factors and achieving precise spatial and temporal pharmacodynamic control, thereby significantly reducing the toxic burden on normal tissues. Furthermore, the prodrug's stable structure, ease of assembly, and high controllability facilitate clinical formulation development and large-scale application.

[0079] Depending on the specific treatment needs, the copper content can be adjusted to enhance or weaken the copper death effect, thus balancing treatment intensity and safety. This prodrug can also be used in combination with radiotherapy, immune checkpoint inhibitors, and other treatments to further improve efficacy. In extreme resistance models where temozolomide is completely ineffective, a copper-metformin prodrug can be constructed without temozolomide, utilizing both copper death and metabolic interference mechanisms to independently exert its antitumor effect. Regarding drug formulation, the prodrug can be further developed into injectable, implantable, or targeted delivery systems, such as by surface modification with tumor-targeting ligands (e.g., RGD peptides) to improve localization efficiency.

[0080] Preferably, in the above-mentioned uses, such as the copper / temozolomide / metformin prodrug used in the glutathione-responsive release of temozolomide and copper ions for the synergistic treatment of glioma, the prodrug depolymerization is triggered by glutathione in the tumor microenvironment, releasing the active ingredient to enhance the therapeutic effect.

[0081] This implementation method utilizes the characteristic that the glutathione (GSH) concentration in the glioma microenvironment is significantly higher than that in normal tissue, and designs a GSH-responsive copper / temozolomide / metformin three-component prodrug. Upon entering the tumor tissue, the copper coordination bonds in the prodrug structure are disrupted by the high concentration of GSH, causing structural breakage and releasing copper ions and temozolomide into the intracellular environment. The released copper ions induce copper death, disrupting mitochondrial metabolism in tumor cells; temozolomide acts as an alkylating agent, causing DNA damage; and metformin synergistically inhibits energy metabolism in tumor cells. The three components are released on demand and act synergistically in the tumor microenvironment, thereby improving therapeutic selectivity and killing efficiency.

[0082] The GSH responsiveness of the prodrug ensures that its active release is primarily concentrated in tumor tissue, reducing non-specific toxicity to normal tissues and improving treatment safety and efficacy. The synergistic release of copper ions and temozolomide simultaneously acts on the mitochondrial metabolism and DNA structure of tumor cells, interfering with their survival mechanisms through two key pathways. Metformin further regulates energy metabolism by activating the AMPK pathway, achieving a triple attack. Furthermore, this release mechanism provides a reliable basis for controlled-release strategies; the drug release process is time- and concentration-dependent, which helps maintain sustained efficacy and reduces the frequency of dosing. This mechanism is particularly suitable for diffuse gliomas that are difficult to completely surgically remove, improving the local concentration of drug efficacy.

[0083] Glutathione-responsive structures can achieve response rate control by adjusting the molar ratio of copper to drug, prodrug particle size, or coordination bond type. In different tumor types, such as those with low GSH concentrations, multi-stimulation responsive structures, such as acidic or enzyme-sensitive triggering mechanisms, can be designed to replace or supplement GSH responsiveness. Furthermore, the prodrug surface can be modified with PEG or targeting ligands to improve its circulation time and tissue specificity in vivo. The ratio of the three components can also be adjusted according to the lesion type; for example, in hypermetabolic tumors, increasing the metformin ratio can enhance the metabolic interference effect. Structurally, encapsulation methods such as microcapsules and liposomes can further delay release and improve tissue penetration.

[0084] To more clearly illustrate the technical solution and beneficial effects of the present invention, the following detailed description is provided in conjunction with specific embodiments. The embodiments of the present invention are generally divided into three levels: First, Examples 1 to 7 mainly describe the preparation process parameters, structural characterization, and condition control of the copper / temozolomide prodrug to verify the feasibility and responsive characteristics of the two-component prodrug; second, Examples 8 to 10 further introduce metformin into the copper / temozolomide prodrug to construct a three-component prodrug, and explore its assembly mechanism and structural stability; finally, Examples 11 to 13 focus on the glutathione responsiveness, drug release behavior, and cytotoxicity evaluation of the three-component prodrug, thereby verifying its practical application potential in the treatment of drug-resistant glioblastoma. It should be understood that the following embodiments are only for illustrating the present invention and are not intended to limit the scope of protection of the present invention.

[0085] Example 1: Preparation and characterization of copper / temozolomide prodrug In this embodiment, a method for preparing a copper / temozolomide prodrug is provided. The specific steps are as follows: Under 40°C water bath conditions, 1.5 mL of temozolomide (TMZ) aqueous solution with a concentration of 4 mg / mL was added to 30 mL of deionized water. After stirring until homogeneous, 90 μL of copper nitrate trihydrate (Cu(NO3)2·3H2O) aqueous solution with a concentration of 241.6 mg / mL was slowly added dropwise. After continued stirring, 60 μL of sodium hydroxide (NaOH) aqueous solution with a concentration of 40 mg / mL was added to adjust the pH. The mixture was stirred continuously at a constant temperature for 4 hours.

[0086] After the reaction was completed, the reaction solution was centrifuged at 10,000 g for 10 minutes. After removing the supernatant, the lower precipitate was resuspended in deionized water to obtain an aqueous solution of the copper / temozolomide prodrug. This aqueous solution can be used directly for characterization and analysis, or the prodrug solid powder can be obtained by freeze-drying.

[0087] To verify the morphology and structure of the obtained product, the prodrug sample was characterized as follows: Transmission electron microscopy (TEM) characterization (e.g.) Figure 1As shown in the figure: the copper / temozolomide prodrug exhibits a uniform quasi-spherical structure with an average particle size of about 8-12 nanometers and a concentrated particle size distribution, indicating that the prodrug structure has good nanoscale and stability.

[0088] X-ray diffraction (XRD) analysis (such as...) Figure 2 As shown in the diagram): Compared with the temozolomide monomer, the diffraction pattern of the copper / temozolomide prodrug showed no obvious crystalline diffraction peaks, exhibiting amorphous characteristics, indicating that copper and temozolomide formed a new amorphous nanostructure.

[0089] Ultraviolet-Vis absorption spectroscopy (UV-Vis) analysis (e.g.) Figure 3 As shown in the figure): A new absorption peak was observed at 355 nm in the copper / temozolomide prodrug, which is a red shift compared to the temozolomide monomer and copper nitrate solution, indicating that there is a significant coordination effect between copper and temozolomide.

[0090] Fourier transform infrared spectroscopy (FTIR) analysis (such as...) Figure 4 As shown): the prodrug is at 3500~3100 cm. -1 and 1598 cm -1 An N–H related vibrational peak appears at this point, and the C=O stretching vibration changes from 1674 cm⁻¹. -1 Redshifted to 1638 cm -1 The vibrational peak of C=N also shifted, further confirming that a coordinate bond was formed between copper ions and temozolomide.

[0091] X-ray photoelectron spectroscopy (XPS) analysis (such as...) Figure 5 As shown): Cu 2p spectrum shows that copper in the prodrug exists in the form of divalent copper Cu(II). Combined with the peak information of elements such as O, N, and C, it further supports that copper ions and drugs form a stable coordination complex structure.

[0092] This embodiment demonstrates that the coordination self-assembly of temozolomide and copper ions can be achieved under simple aqueous reaction conditions, resulting in a well-defined, nanoscale, and highly responsive copper / temozolomide prodrug, which lays the foundation for the subsequent construction of a multifunctional synergistic therapeutic system.

[0093] Example 2: Preparation of copper / temozolomide prodrug under higher temperature conditions This embodiment provides a method for preparing copper / temozolomide prodrugs at 60°C, further verifying the effect of temperature on the formation of nanoprodrugs.

[0094] The specific steps are as follows: Under constant temperature conditions of 60°C in a water bath, 1.5 mL of temozolomide aqueous solution with a concentration of 4 mg / mL was added to 30 mL of deionized water. After stirring thoroughly, 90 μL of copper nitrate trihydrate aqueous solution with a concentration of 241.6 mg / mL was slowly added while continuing to stir. Subsequently, 60 μL of sodium hydroxide aqueous solution with a concentration of 40 mg / mL was added. The reaction temperature was maintained at 60°C and stirring was continued for 4 hours.

[0095] After the reaction was completed, the reaction solution was centrifuged at 10000g for 10 minutes, the supernatant was removed, and the lower precipitate was resuspended in deionized water to obtain an aqueous solution of copper / temozolomide prodrug. This solution can be freeze-dried to obtain a solid powder of the prodrug for subsequent characterization.

[0096] Structural and performance characterization: The copper / temozolomide prodrug sample prepared under the reaction conditions (60°C) of Example 2 was structurally characterized according to the method of Example 1, and the results are as follows: Transmission electron microscopy (TEM) images (corresponding) Figure 1 The copper / temozolomide prodrug still exhibits a good spherical morphology, with a slightly increased particle size and an average particle size in the range of 10-14 nanometers. This indicates that increasing the reaction temperature can increase the particle formation rate but slightly increase the particle size.

[0097] X-ray diffraction (XRD) analysis (corresponding to) Figure 2 The prodrug still retains its amorphous characteristics and no obvious crystallization peaks have appeared, indicating that the temperature change has not destroyed the amorphous characteristics of the coordination structure.

[0098] Ultraviolet-Visible Absorption Spectroscopy (UV-Vis) Figure 3 ) and infrared spectrum ( Figure 4 Analysis: Consistent with the results of Example 1, the characteristic absorption peak redshift and the C=O and C=N peak shift were still observed, indicating that the coordination between copper and temozolomide is still effective and temperature does not significantly interfere with the coordination process.

[0099] XPS analysis ( Figure 5 The Cu 2p peak indicates that the copper ion still exists in the Cu(II) state, and the N and O spectra further confirm that its coordination environment has not changed significantly.

[0100] Results analysis: Compared to Example 1, this example involves a reaction at a higher temperature, resulting in a slightly larger prodrug particle size, but it still maintains a nanoscale structure and good morphological uniformity, indicating that the reaction temperature has a regulatory effect on prodrug formation. Appropriately increasing the temperature can accelerate the reaction process, but it needs to be controlled within a range that does not cause damage to the coordination structure.

[0101] Example 3: Preparation of copper / temozolomide prodrug at room temperature In this embodiment, a method for synthesizing copper / temozolomide prodrug at room temperature (approximately 25°C) is provided to verify the mild reaction characteristics and applicability of the preparation process of the present invention.

[0102] The preparation steps are as follows: Under stirring conditions at room temperature, 1.5 mL of temozolomide (TMZ) aqueous solution with a concentration of 4 mg / mL was added to 30 mL of deionized water and mixed thoroughly. Then, 90 μL of copper nitrate trihydrate (Cu(NO3)2·3H2O) aqueous solution with a concentration of 241.6 mg / mL was added, and stirring continued for 2 minutes. Next, 60 μL of sodium hydroxide (NaOH) aqueous solution with a concentration of 40 mg / mL was added to adjust the pH of the reaction. The reaction was continued for 4 hours with stirring at room temperature.

[0103] After the reaction was completed, the system was centrifuged at 10000g for 10 minutes, the supernatant was removed, the precipitate was retained and resuspended in deionized water to obtain an aqueous solution of copper / temozolomide prodrug; the prodrug powder can be further obtained by freeze drying.

[0104] Characterization and Analysis: TEM analysis (see) Figure 1 The prodrug exhibits a nanoscale spherical structure with a particle size distribution slightly smaller than that of the sample obtained under high-temperature conditions. The average particle size is approximately 5-9 nanometers, with regular morphology and good dispersibility.

[0105] XRD analysis ( Figure 2 The diffraction pattern shows that the prodrug still has an amorphous structure, and no characteristic crystalline peaks of copper or temozolomide monomers were observed, indicating that coordination formed a new stable amorphous complex.

[0106] UV-Vis absorption spectrum ( Figure 3 The appearance of a new absorption peak at 355 nm and a long-wavelength tailing phenomenon is consistent with the results under high temperature conditions, confirming that copper and temozolomide form an effective coordination.

[0107] FTIR spectroscopy ( Figure 4 The characteristic absorption peaks such as C=O, C=N, and N–H showed a slight red shift, and the shift pattern of coordination-related peaks was consistent with that in Examples 1 and 2.

[0108] XPS analysis ( Figure 5 Copper exists in the form of Cu(II), and the electronic energy spectrum of O and N elements further confirms the stable coordination structure of copper with temozolomide in the prodrug.

[0109] Summary of results: Example 3 demonstrates that the synthesis of copper / temozolomide prodrugs can be successfully achieved even without heating, through only stirring at room temperature. The obtained prodrug has a stable structure and uniform particle size, indicating that the method of the present invention has good process compatibility and ease of operation, and is suitable for the development of thermosensitive components or green formulation systems.

[0110] Example 4: Preparation of copper / temozolomide prodrug under reduced sodium hydroxide dosage conditions In this embodiment, by reducing the amount of sodium hydroxide added, the relationship between the basic strength required for prodrug formation and the coordination assembly efficiency was examined, further verifying the controllability of the preparation process of the present invention.

[0111] The preparation steps are as follows: Under stirring conditions in a 40°C water bath, 1.5 mL of temozolomide aqueous solution with a concentration of 4 mg / mL was added to 30 mL of deionized water, and 90 μL of copper nitrate trihydrate aqueous solution with a concentration of 241.6 mg / mL was slowly added dropwise. After thorough mixing, 30 μL of sodium hydroxide aqueous solution with a concentration of 40 mg / mL (half the amount of NaOH used in Example 1) was added to adjust the pH value.

[0112] The reaction temperature was maintained at 40℃, and the reaction was stirred for 4 hours. After the reaction was completed, the sample was centrifuged at 10000g for 10 minutes to remove the supernatant. The precipitate was resuspended in deionized water to obtain an aqueous solution of copper / temozolomide prodrug, which could be freeze-dried to obtain a solid powder of the prodrug.

[0113] Analysis and characterization results: TEM image (corresponding) Figure 1 The obtained prodrug still exhibits a spherical nanostructure with a particle size slightly larger than that of Example 1, approximately 10-16 nm. The particle size distribution is uniform, indicating that even with a reduction in the amount of sodium hydroxide, nanoparticles can still be stably formed.

[0114] XRD analysis ( Figure 2 The spectrum showed no obvious crystallization peaks, maintaining an amorphous structure, indicating that the low NaOH dosage did not interfere with the formation of the amorphous prodrug structure.

[0115] UV-Vis spectrum ( Figure 3 The characteristic absorption peak at 355 nm was still observed, and the redshift phenomenon still existed, indicating that the coordination relationship between copper ions and temozolomide remained effective under these conditions.

[0116] FTIR spectroscopy ( Figure 4 The functional groups such as C=O, C=N, and NH still show a shift in the position of the characteristic absorption peak, which is similar to the results under high NaOH conditions, further verifying the effectiveness of the coordination structure.

[0117] XPS analysis ( Figure 5Copper exists in the form of Cu(II), and no Cu(0) or Cu(I) peaks were observed, indicating that the copper ion oxidation state in the prodrug is stable.

[0118] Example 4 shows that the assembly of copper / temozolomide prodrug can still be successfully achieved under lower alkaline conditions (half the amount of NaOH), indicating that the preparation method of the present invention has advantages such as high environmental tolerance and strong adjustability of reaction conditions, and is suitable for the development of different types of formulations, which helps to improve process flexibility and product consistency.

[0119] Example 5: Preparation of copper / temozolomide prodrug under reduced copper ion feed conditions In this embodiment, we explore whether the prodrug can still be stably formed through coordination self-assembly when the amount of copper source is reduced, and examine the changes in its morphology and structural characteristics.

[0120] The preparation steps are as follows: Under stirring conditions in a 40°C water bath, 1.5 mL of temozolomide aqueous solution (4 mg / mL) was added to 30 mL of deionized water. After stirring until homogeneous, 60 μL of copper nitrate trihydrate aqueous solution (241.6 mg / mL) was added (the amount of copper source was reduced by one-third compared to Example 1). Subsequently, 60 μL of sodium hydroxide aqueous solution (40 mg / mL) was added to adjust the pH. The mixed reaction system was stirred continuously for 4 hours.

[0121] After the reaction was complete, the supernatant was removed by centrifugation at 10000g for 10 minutes. The precipitate was resuspended in deionized water to obtain an aqueous solution of copper / temozolomide prodrug. The sample could be freeze-dried to obtain a powder for further analysis.

[0122] Structural and performance analysis: TEM images ( Figure 1 The particle size is slightly smaller, concentrated in the range of 6~10nm, indicating that although the amount of copper ions is reduced, the coordination self-assembly process can still proceed smoothly, and the resulting nanoparticles have a compact structure and good dispersion.

[0123] XRD patterns ( Figure 2 The amorphous characteristics were maintained, and no characteristic peaks of metallic copper or temozolomide crystals were observed, indicating that copper ions still successfully participated in the construction of the coordination structure.

[0124] Ultraviolet-Visible Absorption Spectroscopy (UV-Vis) Figure 3 The 355nm absorption peak still exists, but its intensity is slightly lower than that in Example 1, indicating that the copper-temozolomide coordination formation is still effective, only slightly weaker, which is in line with the expectation of reducing the amount of copper.

[0125] FTIR analysis ( Figure 4The C=O and C=N stretching vibrations still exhibit redshift, further verifying that the coordination relationship still exists and the structure has not undergone any fundamental change.

[0126] XPS ( Figure 5 The copper still exists in the form of Cu(II) and no reduction products are formed, indicating that the copper in the prodrug maintains chemical stability and has not been degraded or deactivated.

[0127] Summary of results: This embodiment verifies that even with a reduced proportion of copper source, a copper / temozolomide prodrug can still be successfully constructed, indicating that the system has strong adaptability within the copper ion concentration window and can flexibly adjust the amount of copper added according to the target toxicity requirements, thereby achieving a balance between efficacy and toxic side effects.

[0128] Example 6: Preparation of copper / temozolomide prodrug under conditions of further reduced copper source usage This embodiment aims to control the copper ion dosage ratio to a lower level (only 1 / 3 of that in Example 1) to evaluate the impact of the lower limit of copper dosage on the self-assembly, structural integrity and functional retention of nanoprodrugs, and to provide a theoretical basis for the control of copper ion toxicity in clinical applications.

[0129] The preparation steps are as follows: Under stirring conditions in a 40°C water bath, 1.5 mL of temozolomide aqueous solution with a concentration of 4 mg / mL was added to 30 mL of deionized water, followed by the slow addition of 30 μL of copper nitrate trihydrate aqueous solution with a concentration of 241.6 mg / mL (one-third of the copper dosage in Example 1). After stirring until homogeneous, 60 μL of sodium hydroxide aqueous solution with a concentration of 40 mg / mL was added.

[0130] The reaction temperature was maintained at 40℃, and the mixture was stirred continuously for 4 hours. The supernatant was then removed by centrifugation at 10000g for 10 minutes. The resulting precipitate was resuspended in deionized water to obtain an aqueous solution of the copper / temozolomide prodrug. A solid powder sample could then be obtained by freeze-drying.

[0131] Structural characterization and analysis: TEM analysis (see) Figure 1 The prodrug still exhibits a spherical nanostructure with a particle size between 6 and 9 nm. It has good dispersibility and no obvious agglomeration phenomenon was observed, indicating that even if the amount of copper added is greatly reduced, it can still drive the formation of a nano prodrug structure.

[0132] XRD patterns ( Figure 2 ): It retains amorphous characteristics, and no characteristic diffraction peaks of copper ions or temozolomide crystals are observed, indicating a stable structure.

[0133] UV-Vis spectrum ( Figure 3The characteristic absorption peak at 355 nm is still visible, but its intensity is slightly lower than that of Examples 1 and 5, which is consistent with the characteristic expression after the copper coordination ratio is reduced.

[0134] FTIR spectra ( Figure 4 C=O stretching vibration from 1674 cm -1 Redshifted to 1640 cm -1 The C=N vibration region peak shifted slightly, which still indicates the presence of copper-drug coordination bonds and that the prodrug assembly characteristics have not been lost.

[0135] XPS analysis ( Figure 5 ): It was confirmed that copper still exists in the prodrug structure in the form of Cu(II), without any change in valence state or reduction inactivation, supporting the stable existence of copper under low-dose conditions.

[0136] This embodiment verifies that even when the proportion of copper ions is reduced to 1 / 3 of the original amount, copper / temozolomide prodrug nanoparticles with well-defined structures and controllable particle sizes can still be stably formed, and they still have typical coordination absorption peaks and structural characteristics. This indicates that the preparation system has good lower limit process adaptability in terms of copper dosage control, which helps to further control the risk of copper-related toxicity while ensuring efficacy.

[0137] Example 7: Preparation of copper / temozolomide prodrug under extended stirring time conditions This embodiment aims to investigate whether extending the stirring time can further improve the structural integrity, crystallinity changes, or particle size uniformity of the copper / temozolomide prodrug under the condition of constant temperature and reaction system, so as to provide data support for optimizing the reaction time.

[0138] The preparation steps are as follows: Under constant temperature conditions (40℃ water bath), 1.5 mL of temozolomide (TMZ) aqueous solution (4 mg / mL) was added to 30 mL of deionized water. After thorough mixing, 90 μL of copper nitrate trihydrate aqueous solution (241.6 mg / mL) was added, and the mixture was stirred for 1 minute. Then, 60 μL of sodium hydroxide aqueous solution (40 mg / mL) was added. The stirring time was extended to 10 hours, while maintaining a constant temperature.

[0139] After the reaction was complete, the sample was centrifuged at 10000g for 10 minutes to remove the supernatant. The precipitate was resuspended in deionized water to obtain a copper / temozolomide prodrug aqueous solution, which was then freeze-dried to obtain a prodrug solid powder sample.

[0140] Structural and performance analysis: TEM images ( Figure 1The prodrug still maintains a quasi-spherical morphology with a concentrated particle size distribution of approximately 10-14 nm. Compared with Example 1, the particle size is slightly larger but more uniform, indicating that extending the reaction time promotes the self-assembly maturation process of the prodrug structure.

[0141] XRD analysis ( Figure 2 The result still shows an amorphous structure and no new crystal peaks appear, indicating that prolonged stirring will not cause crystal precipitation or spontaneous rearrangement.

[0142] UV-Vis absorption spectrum ( Figure 3 The absorption peak at 355 nm is clearer and its intensity increases in a shorter time, indicating that the coordination degree between copper and temozolomide is deepened and the coordination stability is improved.

[0143] FTIR spectroscopy ( Figure 4 The redshift of the vibrational peaks related to C=O and C=N is slightly larger, indicating that coordination interactions tend to be more complete under long-term reaction conditions.

[0144] XPS analysis ( Figure 5 The Cu 2p peak intensity is stable, and there is no Cu. 0 The presence of the signal indicates that the copper ions were not reduced during the long-term stirring process and remained stably present in the prodrug structure as Cu(II).

[0145] This embodiment demonstrates that extending the reaction time from 4 hours to 10 hours at 40°C enhances the coordination efficiency and structural stability of copper and temozolomide, resulting in more uniform prodrug particle size, more significant coordination absorption, and a more pronounced red shift of functional groups, without inducing adverse side reactions such as crystallization, aggregation, or copper reduction. Therefore, stirring time is one of the effective parameters for controlling the degree of assembly and is suitable for nanomedicine development scenarios with high requirements for structural uniformity.

[0146] Example 8: Preparation of a copper / temozolomide / metformin three-component prodrug This embodiment aims to introduce the metabolic regulator metformin into the copper / temozolomide prodrug, and construct a three-component nanoprodrug system (Cu-TMZ-MET) by coordinating metformin with the copper ions already in the system. This will endow the prodrug with additional anti-tumor mechanisms (such as AMPK activation, metabolic inhibition, etc.) and enhance the comprehensive therapeutic effect on glioma.

[0147] The preparation steps are as follows: Under stirring conditions at room temperature, 2 mL of a 3 mg / mL copper / temozolomide prodrug solution was added to 28 mL of deionized water. Subsequently, 600 μL of a 20 mg / mL metformin solution was added. The reaction system was stirred continuously at room temperature for 2 hours to promote the coordination of copper ions with metformin.

[0148] After the reaction was complete, the sample was centrifuged at 10000g for 10 minutes to remove the supernatant. The precipitate was then resuspended in deionized water to obtain an aqueous solution of copper / temozolomide / metformin prodrug. This solution was freeze-dried to obtain a three-component prodrug powder for subsequent characterization and bioactivity evaluation.

[0149] Structural characterization and analysis: TEM images ( Figure 6 The prodrug exhibits a regular spherical nanostructure with an average particle size of approximately 31.9 nm, which is significantly larger than that of the copper / temozolomide dual-component prodrug (~10 nm). This indicates that the introduction of metformin increased the structural complexity and particle size of the prodrug.

[0150] Ultraviolet-Visible Absorption Spectroscopy (UV-Vis) Figure 7 Compared to the copper / temozolomide prodrug, the three-component prodrug exhibits a new absorption peak at approximately 232 nm, corresponding to the characteristic absorption of metformin, verifying its successful introduction into the prodrug structure; the 355 nm absorption peak is still retained, indicating that the coordination relationship between copper and temozolomide has not been disrupted.

[0151] Interaction mechanism analysis Figure 8 a~c): Figure 8 a shows the changes in the absorption spectra of the three-component prodrug under different interfering agents, indicating that its structural assembly involves copper coordination, electrostatic interaction and hydrophobic interaction; Figure 8 b represents the particle size distribution measured by DLS, which changes significantly under the influence of different disruptive agents, further verifying the composite mechanism; Figure 8 c is a TEM image under destructive effects, showing that the structure gradually decomposes from intact nanospheres, indicating that the prodrug is a typical multiphase coordination-driven nanoassembly system.

[0152] This embodiment successfully constructed a three-component copper / temozolomide / metformin nanoprodrug structure. It not only maintained the nanomorphology and coordination structure of the copper / temozolomide prodrug but also effectively introduced metformin, providing the prodrug with a synergistic anti-tumor mechanism and controllable particle size regulation capability. This three-component prodrug holds promise for achieving combined therapy through copper death, DNA damage, and metabolic interference mechanisms, offering a more effective treatment for multidrug-resistant tumors.

[0153] Example 9: Preparation of copper / temozolomide / metformin prodrug under extended reaction time conditions This embodiment aims to evaluate whether extending the stirring time during the self-assembly reaction of copper / temozolomide and metformin improves the assembly degree, particle size uniformity and structural integrity of the three-component nanoprodrug, providing parameter references for optimizing the preparation process.

[0154] The preparation steps are as follows: Under stirring conditions at room temperature, 2 mL of a 3 mg / mL copper / temozolomide prodrug solution was added to 28 mL of deionized water. This was followed by the addition of 600 μL of a 20 mg / mL metformin solution.

[0155] Unlike Example 8, this example extends the stirring reaction time to 3 hours to ensure that the coordination and self-assembly processes are fully carried out.

[0156] After the reaction was completed, the supernatant was removed by centrifugation at 10000g for 10 minutes. The precipitate was resuspended in deionized water to obtain an aqueous solution of copper / temozolomide / metformin prodrug, which could be freeze-dried to obtain a solid powder sample.

[0157] Structural analysis and characterization: TEM images ( Figure 6 The prodrug obtained after extending the reaction time has a more uniform structure, with the particle size controlled between 30 and 32 nm. It has a good spherical morphology, clear boundaries, and no obvious aggregation or collapse, indicating that the structure is more compact and stable.

[0158] Ultraviolet-Visible Absorption Spectroscopy (UV-Vis) Figure 7 The signal intensity of the characteristic absorption peaks at 232 nm and 355 nm is further enhanced, indicating that the coordination relationship between metformin and copper, and between copper and temozolomide is more stable.

[0159] Interaction destruction experiment ( Figure 8 a~c): Figure 8 a (absorption spectrum): Compared with Example 8, the structure has enhanced resistance to external disturbances; Figure 8 b (DLS particle size analysis): The more concentrated particle size distribution indicates a more compact assembly structure; Figure 8 c(TEM): The particle morphology is maintained for a longer time after treatment with the destructive agent, and the resistance to dissociation is improved.

[0160] This embodiment demonstrates that appropriately extending the reaction time of the copper / temozolomide / metformin triad prodrug can further improve its structural stability and particle size uniformity, contributing to the formation of denser and more regular nanoparticles and enhancing its stable release capability and controlled release performance under complex in vivo environments. This has significant process value for subsequent in vivo delivery, safety evaluation, and targeted modification.

[0161] Example 10: Preparation of copper / temozolomide / metformin prodrug with reduced metformin feed ratio The study investigated whether reducing the amount of metformin added affected the nanostructure formation, coordination stability, and absorption characteristics of the prodrug while keeping other reaction conditions constant, thereby evaluating the structural role and dosage control range of metformin in the three-component prodrug.

[0162] The preparation steps are as follows: Under stirring conditions at room temperature, 2 mL of a 3 mg / mL copper / temozolomide prodrug solution was added to 30 mL of deionized water. Then, 300 μL of a 20 mg / mL metformin solution was added (half the 600 μL in Example 8).

[0163] The reaction was stirred for 2 hours under constant temperature conditions to allow copper ions to fully coordinate with metformin and complete the three-component assembly.

[0164] After the reaction was completed, the supernatant was removed by centrifugation at 10000g for 10 minutes. The precipitate was then resuspended in deionized water to obtain an aqueous solution of copper / temozolomide / metformin prodrug, which can be further freeze-dried to obtain a powder sample for subsequent analysis.

[0165] Structural characterization and analysis: TEM images ( Figure 6 The particle morphology remains quasi-spherical, with an average particle size slightly smaller than that of Examples 8 and 9, approximately 25-30 nm. This indicates that as the metformin concentration decreases, the assembly density decreases and the particle size decreases slightly, but the nanostructure is still maintained.

[0166] UV-Vis absorption spectrum ( Figure 7 The characteristic absorption peak of metformin at 232 nm is still visible, but its intensity is significantly lower than that in Examples 8 and 9, indicating that the number of metformin involved in coordination is reduced and the degree of coordination is weakened; the copper-TMZ coordination peak at 355 nm is not significantly affected.

[0167] DLS dynamic light scattering data ( Figure 8 b): The widening of the particle size distribution indicates that the compactness of the assembled structure has decreased slightly, but it still remains basically stable.

[0168] TEM morphology image ( Figure 8 c): The structure is intact, but its resistance to damage is slightly lower than that of Example 9. It is more prone to particle deformation or loosening under strong interference conditions.

[0169] This embodiment demonstrates that when the amount of metformin is reduced to 50% of the original amount, the copper / temozolomide / metformin three-component prodrug can still successfully form a stable nanostructure, possessing basic coordination and self-assembly capabilities. Although the reduced involvement of metformin slightly decreases the prodrug structure density and particle size, the overall structure is still maintained, and the coordination absorption characteristics remain. This indicates that the present invention has good ratio adjustability, which is beneficial for flexibly adjusting the dosage ratio of metabolic inhibitors to meet different therapeutic needs.

[0170] Example 11: Fluorescence response test of copper / temozolomide / metformin prodrug under glutathione conditions This embodiment aims to investigate the response performance of copper / temozolomide / metformin nanoprodrugs under the action of glutathione (GSH), a tumor reducing environment mimic. The ability of the prodrugs to consume GSH was detected by the fluorescent probe o-phthalaldehyde (OPA), thereby indirectly verifying the disintegration of the prodrug structure in the presence of GSH.

[0171] Experimental materials and conditions: Copper / temozolomide / metformin prodrug: prepared from Example 8; Glutathione (GSH) concentration: 0.5 mM; Fluorescent indicator: orthophthalaldehyde (OPA); Prodrug concentration gradient: 0, 10, 20, 30, 40, 50, 60, 70, 80 μg / mL; Excitation wavelength: 350nm; Emission wavelength range: 350~650nm; Testing instrument: Fluorescence spectrophotometer.

[0172] Experimental steps: 1. Prepare copper / temozolomide / metformin prodrug solutions of different concentrations according to the set concentration gradient; 2. Add a fixed concentration of GSH solution (0.5 mM) to each group of samples; 3. Add OPA solution, mix well, and react at room temperature in the dark for 30 minutes; 4. Use a fluorescence spectrophotometer to perform scanning measurements and record the effect of different concentrations of prodrug on fluorescence intensity.

[0173] Experimental results (corresponding) Figure 9 ): Figure 9 The results showed that as the concentration of the prodrug gradually increased, the fluorescence intensity gradually decreased, exhibiting a significant concentration-dependent inhibition effect. This indicates that the prodrug can react with GSH, consuming glutathione in the system and thus inhibiting the fluorescence reaction between OPA and GSH; The fluorescence inhibition trend was negatively correlated with the prodrug concentration, indicating that the prodrug has good responsiveness and consumption capacity to GSH.

[0174] Example 11 demonstrates that the copper / temozolomide / metformin prodrug constructed in this invention exhibits excellent glutathione-responsive properties. In a simulated tumor microenvironment, the prodrug rapidly consumes GSH, triggering the depolymerization of the nanostructure and releasing the drug component. This responsiveness provides important mechanistic support for the prodrug's "tumor microenvironment-triggered release," contributing to improved therapeutic selectivity and reduced systemic toxicity.

[0175] Example 12: Temozolomide release experiment of copper / temozolomide / metformin prodrug under glutathione conditions By simulating the highly reducing environment of tumors in vivo, the drug release behavior and release kinetics of the three-component nanoprodrug of this invention in the presence of glutathione were investigated, clarifying its depolymerization ability under the action of GSH and its ability to effectively release temozolomide.

[0176] Experimental materials and conditions: Test sample: Copper / temozolomide / metformin prodrug prepared in Example 8; Simulated environment: 2mM glutathione solution (approximate to the GSH concentration in tumor cells); Dialysis bag specifications: molecular weight cutoff 3000 Da; Time points: 0, 5 min, 15 min, 30 min, 45 min, 1 h, 1.5 h, 2 h, 3 h, 4 h, 5 h, 7 h; Detection method: Ultraviolet-visible spectrophotometry, detection wavelength 254nm (TMZ characteristic absorption peak); Testing instrument: Ultraviolet-visible spectrophotometer.

[0177] Experimental steps: 1. Disperse a certain amount of prodrug sample in a 2 mM GSH solution; 2. Pack into a dialysis bag with a molecular weight cutoff of 3000 Da; 3. Place the dialysis bag in 60 mL of GSH solution and shake continuously using a shaker; 4. Take out the dialysate sample at the preset time point and measure its absorbance value at 254 nm; 5. Based on the pre-established standard curve (see...) Figure 12 (b) Calculate the concentration of temozolomide released; 6. Calculate the cumulative release at each time point and plot the release curve (see...). Figure 12 c).

[0178] Experimental Results and Analysis: Figure 12a: Standard UV absorption curves of temozolomide at different concentrations are shown; Figure 12 b: At a wavelength of 254 nm, the absorbance of temozolomide showed a good linear relationship with its concentration (R... 2 =0.99653); Figure 12 c: In the presence of GSH, the release of temozolomide gradually increases over time, with the release rate being faster in the first 2 hours and then plateauing between 4 and 7 hours. Compared with the control group without GSH, the GSH-treated group had a significantly higher release rate, indicating that GSH triggered the depolymerization of the prodrug structure and significantly promoted the release of temozolomide.

[0179] This embodiment verifies that the copper / temozolomide / metformin prodrug exhibits typical glutathione-responsive release characteristics. In the high GSH environment of tumors, it can rapidly deconstruct its nanostructure, releasing active temozolomide drug molecules, realizing a smart drug delivery strategy of "tumor microenvironment-triggered controlled release," providing strong mechanistic support for targeted anti-tumor therapy.

[0180] Example 13: In vitro toxicity test of copper / temozolomide prodrug against U87 / TR glioma cells This experiment aims to evaluate the cytotoxic effects of copper / temozolomide prodrug and copper / temozolomide / metformin triad prodrug on temozolomide-resistant glioblastoma cells (U87 / TR) in vitro, compare their inhibitory capacity and concentration dependence, and provide an experimental basis for verifying their mechanism of reversing drug resistance and synergistic antitumor action.

[0181] Experimental materials and conditions: Cell line: U87 / TR (TMZ-resistant human glioma cells); Culture plate: 96-well cell culture plate; Initial inoculation density: 1×10 4 cells / pores; Processing time: 24 hours of prodrug incubation; Assay method: CCK-8 (cell counting kit); Prodrug treatment concentrations: 0, 10, 20, 30, 40, 50, 60, 70, 80 μg / mL; Detection wavelength: 450nm; Instrument: Microplate reader; Control group: No drug treatment (0 μg / mL).

[0182] Experimental steps: 1. Arrange U87 / TR cells at a density of 1×10⁶ cells per well. 4The samples were seeded at a density in 96-well plates and incubated in a CO2 incubator (37°C, 5% CO2) for 24 hours. 2. Replace the culture medium with different concentrations of copper / temozolomide prodrug or copper / temozolomide / metformin prodrug, and continue incubation for 24 hours; 3. Remove the culture medium, add 10 μL of CCK-8 medium to each well, and continue incubation for 1 hour; 4. Use an ELISA reader to measure the absorbance at 450 nm and calculate the relative cell viability; 5. The results were plotted as a curve showing the relationship between cell activity and drug concentration (see...). Figure 13 ).

[0183] Experimental Results and Analysis (corresponding) Figure 13 ): Figure 13 The results showed that as the drug concentration increased, the survival rate of U87 / TR cells decreased significantly, exhibiting a typical dose-dependent inhibition curve; The cytotoxic effect of the copper / temozolomide / metformin three-component prodrug was significantly better than that of the copper / temozolomide two-component prodrug; Within the concentration range of 50–80 μg / mL, the three-component prodrug can reduce cell viability to below 50%, exhibiting a significant synergistic toxicity enhancement effect; The cell viability in the control group remained above 90%, indicating that the efficacy of the drug did indeed originate from the prodrug action.

[0184] This embodiment clearly demonstrates that both the copper / temozolomide prodrug and the copper / temozolomide / metformin prodrug constructed in this invention can produce significant toxic effects on TMZ-resistant U87 / TR cells. In particular, the three-component prodrug exhibits a stronger synergistic antitumor ability, indicating that the prodrug not only retains the chemotherapeutic activity of temozolomide, but also enhances its killing effect on drug-resistant cells through copper death and metabolic interference mechanisms, providing a new strategy for solving the clinical drug resistance problem of TMZ.

[0185] Through Examples 1 to 13 above, this invention systematically demonstrates a method for constructing copper / temozolomide and copper / temozolomide / metformin nanoprodrugs and their performance regulation strategies. Examples 1 to 7 focus on optimizing the preparation process parameters of the two-component prodrug (Cu–TMZ), verifying that the prodrug can be stably assembled under different temperatures (room temperature to 60°C), stirring times (4–10 hours), copper source amounts (standard to 1 / 3 of the amount), and alkaline conditions (NaOH halved), indicating that the method possesses good process adaptability and controllability.

[0186] Examples 8 to 10 were extended to the construction of a three-component prodrug (Cu-TMZ-MET), further incorporating metformin into the assembly process. The resulting prodrug particle size increased (to approximately 30 nm), and its structural stability and assembly mechanism were confirmed using multiple characterization techniques including TEM, UV, FTIR, and XPS, involving multiple driving factors such as copper coordination, electrostatic interactions, and hydrophobic interactions. The three-component system exhibited higher structural compactness and functional synergistic potential.

[0187] Examples 11 to 13 functionally validated the glutathione responsiveness of the nanoprodrug of the present invention. Figure 9 ), controllable release capability of temozolomide ( Figure 12 And its killing effect on TMZ-resistant glioma cells (U87 / TR) Figure 13 The results all indicate that the prodrug has significant tumor microenvironment responsiveness and the potential to reverse drug resistance.

[0188] In summary, the nanoprodrugs provided by this invention have multiple advantages, including simple structure, mild preparation conditions, biodegradability, strong responsiveness, and synergistic antitumor effects. They have broad application prospects in the treatment of glioblastoma, especially drug-resistant cases, and possess significant clinical and industrial value.

[0189] It should be understood that although this specification has described the invention in detail with reference to specific embodiments, the above embodiments are only used to illustrate the technical principles and core solutions of the invention and do not constitute a limitation on the scope of protection of the invention. Equivalent substitutions or modifications made by those skilled in the art to the structure, composition, proportions, process parameters, etc., of the invention without departing from the spirit and substance of the invention should all be covered within the scope of protection of the invention.

[0190] In particular, the component ratio, particle size control, reaction time, temperature conditions, coordination order, etc. of the copper / temozolomide prodrug and the copper / temozolomide / metformin prodrug involved in this invention can be appropriately adjusted according to different clinical or industrialization needs; as long as it does not deviate from the technical essence of this invention and the technical problem it solves, it should be considered to fall within the scope of this invention.

[0191] Therefore, the scope of protection of this invention should be determined by the content defined in the appended claims, while the specific technical content in the specification and embodiments can be used as the basis for interpreting the claims.

Claims

1. A copper / temozolomide prodrug, characterized in that, The prodrug is formed by the self-assembly of temozolomide and copper ions through coordination, wherein: Temozolomide has a mass fraction of 50%~90%. The mass fraction of copper ions is 10%~50%. The prodrug is glutathione responsive.

2. The copper / temozolomide prodrug according to claim 1, characterized in that, The copper ions are divalent copper ions.

3. A copper / temozolomide / metformin prodrug, characterized in that, It is formed by coordination and assembly of temozolomide, copper ions, and metformin, wherein: Temozolomide has a mass fraction of 20%~60%. The mass fraction of copper ions is 20%~40%. Metformin has a mass fraction of 1% to 60%. The prodrug is glutathione responsive.

4. The copper / temozolomide / metformin prodrug according to claim 3, characterized in that, Metformin is directly added to the copper / temozolomide prodrug solution, where a coordination reaction occurs with the presence of existing copper ions, forming a three-component prodrug structure.

5. A method for preparing the prodrug according to claim 1 or 3, characterized in that, Includes the following steps: Add temozolomide aqueous solution to 10-120 mL of water. Add a divalent copper salt aqueous solution, Add sodium hydroxide aqueous solution, stir and react for 10 minutes to 24 hours, at a temperature of room temperature to 60°C. After the reaction was completed, the sample was centrifuged, the precipitate was resuspended, and an aqueous solution of copper / temozolomide prodrug was obtained. It can be further dried to obtain a copper / temozolomide prodrug solid powder.

6. The preparation method according to claim 5, characterized in that, The divalent copper salt is one of copper chloride, copper nitrate, or copper sulfate; The concentration of temozolomide is 1-5 mg / mL. The concentration of divalent copper salts is 50~800 mg / mL. The concentration of sodium hydroxide is 0.01~40 mg / mL. The molar ratio of divalent copper salt to temozolomide is 0.1~5:

1.

7. A method for preparing the copper / temozolomide / metformin prodrug according to claim 3, characterized in that, Includes the following steps: Add copper / temozolomide prodrug to water. Add metformin aqueous solution, stir for 20 minutes to 24 hours, and maintain the reaction temperature at room temperature. After centrifugation, the precipitate was resuspended to obtain an aqueous solution of copper / temozolomide / metformin prodrug. It can be further dried to obtain a copper / temozolomide / metformin prodrug solid powder.

8. The preparation method according to claim 7, characterized in that, The molar ratio of copper / temozolomide prodrug to metformin is 0.1–10:

1. The concentration of metformin is 0.1~50 mg / mL.

9. The use of a prodrug as described in any one of claims 1 to 4 in the preparation of a drug for treating drug-resistant glioblastoma, characterized in that, The prodrug has a toxic effect on temozolomide-resistant glioblastoma cells through a copper death mechanism.

10. A copper / temozolomide / metformin prodrug as described in claim 3 or 4, used in the glutathione-responsive release of temozolomide and copper ions for the synergistic treatment of glioma, characterized in that... Glutathione in the tumor microenvironment triggers prodrug depolymerization, releasing the active ingredient to enhance therapeutic efficacy.

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