Copper-loaded nano self-assembled body, and preparation method and application thereof

Abstract

The application discloses a copper-loaded nano self-assembly and a preparation method and application thereof, and the copper-loaded nano self-assembly is formed by self-assembly of a compound N'-linolenoyl-N-methyl thiobenzoyl hydrazine or a pharmaceutically acceptable salt thereof after coordination with copper ions. The N'-linolenoyl-N-methyl thiobenzoyl hydrazine is prepared by acylation reaction of linolenoyl chloride and N-methyl thiobenzoyl hydrazine, and then the N'-linolenoyl-N-methyl thiobenzoyl hydrazine is co-dissolved with DSPE-PEG in an organic solvent and injected into a copper-containing aqueous phase to form nanoparticles by self-assembly. The obtained copper-loaded nano self-assembly has uniform particle size (about 108 nm), is spherical, has good stability, has a significant killing effect on MC38 cells in vitro, can effectively inhibit the growth of a subcutaneous tumor of a mouse in vivo and has good safety. The application overcomes the problems of low solubility and poor copper ion delivery efficiency of existing copper ion carriers, and provides a novel nano delivery platform for copper death-mediated tumor treatment.

Description

Technical Field

[0001] This invention relates to the fields of chemistry and biomedicine, specifically to a novel copper ion carrier, a copper-loaded nano-assembly containing the carrier, a method for preparing the same, and the use of the copper-loaded nano-assembly in the preparation of a drug for treating tumors. Background Technology

[0002] Copper death is a novel form of cell death. Its mechanism involves copper ions directly binding to esterified proteins in the tricarboxylic acid cycle, inducing proteotoxic stress and leading to cell death. This process is independent of classic cell death pathways such as apoptosis, ferroptosis, or necroptosis. This discovery provides a novel strategy for cancer treatment: by increasing the intracellular copper ion concentration in tumor cells, copper death can be specifically induced, thereby achieving effective tumor killing.

[0003] Copper ion carriers are small molecule compounds that can form stable complexes with copper ions and promote their transmembrane transport. Representative drugs include elesclomol and disulfiram. Elsclomol has been used in clinical trials to induce oxidative stress and mitochondrial dysfunction by transporting extracellular copper ions into intracellular spaces. However, existing copper ion carriers have significant limitations: First, most carriers have extremely poor water solubility, making it difficult to maintain effective concentrations under physiological conditions, resulting in low bioavailability; second, due to the lack of targeting or controlled release properties, free copper ion carriers lack selectivity in vivo distribution, easily causing systemic toxicity, and dose-limiting toxic side effects have been observed in clinical applications; third, existing carriers themselves lack sufficient chemical stability, easily degrading or accumulating in the blood, resulting in poor drug-like properties. Therefore, although copper death shows great potential in the field of anti-tumor therapy, how to efficiently and safely deliver copper ions to tumor tissues remains a pressing technical challenge.

[0004] Nanomedicine delivery systems can improve the solubility and pharmacokinetic behavior of poorly soluble drugs, enabling passive or active targeted delivery to tumors and reducing systemic toxicity. In recent years, copper delivery nanoplatforms based on lipids, polymers, or inorganic materials (such as copper-polyphenol nanoparticles) have been reported. However, these systems typically involve complex material synthesis or multi-step preparation processes, and the loading and release behavior of copper ions are difficult to precisely control. Particularly noteworthy is the lack of a single molecule that can both act as a copper ion ligand and possess self-assembly capabilities, thus hindering the one-step construction of copper ion loading and nanostructure formation, limiting the simplification of the preparation process and the improvement of formulation stability. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention designs and synthesizes a novel copper ion carrier—N′-linolenic acid-N-methylthiobenzoylhydrazide. This molecular structure contains a copper coordinating group of thiobenzoylhydrazide and a long-chain linolenic acid lipid tail, enabling it to co-assemble with the auxiliary lipid DSPE-PEG (distearate phosphatidylethanolamine-polyethylene glycol) to form copper-loaded nano-assemblies with uniform particle size and good stability. This system significantly improves the water dispersibility of copper ions and its carrier, enhances antitumor activity, and reduces systemic toxicity, providing an ideal nanodelivery platform for cancer therapy based on copper death mechanisms.

[0006] Therefore, this invention aims to provide a novel copper ion carrier and its copper-loaded nano-self-assembled structure to address the problems of low solubility, poor in vivo delivery efficiency, and insufficient safety of existing copper ion carriers. Specifically, this invention provides a self-assembling copper ion carrier, which is then co-assembled with auxiliary lipids to form copper-loaded nano-self-assembled structures with uniform particle size, good stability, and high antitumor activity.

[0007] To achieve the above objectives, the present invention provides the following technical solution: A copper-loaded nano-self-assembled structure is formed by the self-assembly of N′-linaloyl-N-methylthiobenzoylhydrazine or a pharmaceutically acceptable salt thereof, which is coordinated with copper ions. , (I).

[0008] Furthermore, the copper ions are derived from pharmaceutically acceptable copper salts or solvates.

[0009] Preferably, the copper salt is selected from one or more of copper chloride and copper acetate.

[0010] Furthermore, linolenic acid chloride was subjected to an acylation reaction with N-methylthiobenzoyl hydrazide to prepare N′-linolenic acid-N-methylthiobenzoyl hydrazide.

[0011] The present invention also provides a method for preparing the copper-loaded nano-assemblies: dissolving copper salt in an aqueous phase to obtain a copper-containing aqueous phase; dissolving copper ion carrier and DSPE-PEG in an organic solvent to obtain a first organic solution and a second organic solution, and then mixing the first organic solution and the second organic solution and injecting them into the copper-containing aqueous phase to form copper-loaded nano-assemblies.

[0012] Furthermore, the copper salt is a pharmaceutically acceptable copper salt or a solvation thereof; even further, it is selected from one or more of copper chloride and copper acetate; the organic solvent comprises dimethyl sulfoxide (DMSO).

[0013] Furthermore, the molar ratio of copper ions, copper ion carrier, and DSPE-PEG is 1~50:0.1~1:1~50.

[0014] Finally, this invention provides the use of the copper-loaded nano-assemblies in the preparation of drugs for treating tumors.

[0015] As a preferred embodiment of the present invention, the tumor is colon adenocarcinoma, but it is not limited thereto.

[0016] The beneficial effects of this invention are as follows: (1) The present invention designed and synthesized a novel copper ion carrier N′-linyl-N-methylthiobenzoylhydrazide, which has both copper coordination ability and long-chain lipid properties. It can be co-assembled with DSPE-PEG2000 to form a nano self-assembled body, which significantly improves the water dispersibility and formulation stability of copper ion carrier and copper ions.

[0017] (2) The copper-loaded nano-self-assembled particles of the present invention have an average particle size of about 108 nm, a low PDI (0.16), and are uniformly spherical. The particle size does not change significantly for at least 72 hours at 25°C, showing excellent colloidal stability.

[0018] (3) In vitro cytotoxicity experiments showed that the copper-loaded nano-assemblies exhibited an IC50 effect on MC38 colorectal cancer cells. 50 The value (1.3 μM as copper) is much lower than that of free copper chloride (>1000 μM), and the antitumor activity is increased by more than three orders of magnitude.

[0019] (4) In vivo experiments using the MC38 subcutaneous tumor model in mice demonstrated that intravenous injection of copper-loaded nano-assemblies could significantly inhibit tumor growth without causing a significant decrease in mouse weight, showing good in vivo safety and anti-tumor efficacy, and demonstrating good clinical translation potential. Attached Figure Description

[0020] Figure 1 This is the synthetic route for N′-linolenicoyl-N-methylthiobenzoylhydrazide in Example 1.

[0021] Figure 2 The NMR spectrum of N′-linolenico-N-methylthiobenzoylhydrazide in Example 1 is shown.

[0022] Figure 3 The particle size distribution of the self-assembled structures in Example 2 is shown; where A is a blank nano-self-assembled structure and B is a copper-loaded nano-self-assembled structure.

[0023] Figure 4 This is a transmission electron microscope image of the self-assembled body in Example 2.

[0024] Figure 5The results show the in vitro stability evaluation of the copper-loaded nano-assemblies in Example 3.

[0025] Figure 6 This is an in vitro cytotoxicity experiment of the copper-loaded nano-assemblies in Example 4.

[0026] Figure 7 The effect of copper-loaded nano-self-assembled therapy on tumor growth in Example 5.

[0027] Figure 8 The effect of copper-loaded nano-assemblies on mouse weight in Example 5. Detailed Implementation

[0028] The present invention will be further described below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention.

[0029] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0030] Example 1 Synthesis of N′-linolenico-N-methylthiobenzoylhydrazide The synthetic route of N′-linolenico-N-methylthiobenzoylhydrazide is as follows: Figure 1 As shown, the specific steps are as follows: N-methylthiobenzoyl hydrazide (200 mg, 1.20 mmol, 1.0 eq) and triethylamine (121 mg, 1.20 mmol, 1.0 eq) were weighed and dissolved in 2 mL of anhydrous dichloromethane (DCM), followed by the addition of linolenic acid chloride (360 mg, 1.20 mmol, 1.0 eq). The reaction mixture was stirred at 25 °C for 24 h. After the reaction was complete, the reaction mixture was extracted with DCM. The resulting organic layer was washed successively with 5% citric acid, saturated sodium bicarbonate, and saturated sodium chloride, and then dried over anhydrous sodium sulfate. The solution was purified by silica gel column chromatography using dichloromethane / methanol as the eluent to obtain 376 mg of a yellow solid, with a yield of 73.1%.

[0031] The product 1 H NMR spectrum as shown Figure 2 As shown, it was confirmed that N-methylthiobenzoyl hydrazide and linolenic acid chloride were successfully covalently linked, and the resulting product was high-purity N′-linolenic acid-N-methylthiobenzoyl hydrazide.

[0032] Example 2: Preparation of copper-loaded nano-assemblies 1.0 mg of anhydrous copper chloride (CuCl2) was dissolved in 2 mL of ultrapure water to obtain a copper-containing aqueous phase. 13.7 mg of N′-linolenic acid-N-methylthiobenzoylhydrazide (i.e., the copper ion carrier) was dissolved in 0.1 mL of DMSO to prepare a first solution of 137 mg / mL; 2.3 mg of DSPE-PEG2000 was dissolved in 0.1 mL of DMSO to prepare a second solution of 23 mg / mL.

[0033] The first solution and the second solution were mixed thoroughly at a volume ratio of 1:1 to obtain a mixed organic solution. While stirring, 0.1 mL of this mixed organic solution was slowly injected into the copper-containing aqueous phase. The solution immediately changed from colorless to pale yellow and gradually developed an opalescent appearance. Stirring was continued for 5 minutes to ensure complete assembly.

[0034] As a control, the preparation method of the blank nano-assemblies is as follows: The first solution and the second solution are mixed evenly at a volume ratio of 1:1 to obtain a mixed organic solution. Under stirring, 0.1 mL of the mixed organic solution is slowly injected into the blank aqueous phase, and stirring is continued for 5 min to ensure complete assembly.

[0035] The resulting suspension was transferred to a dialysis bag (molecular weight cutoff 3500 Da) and dialyzed in ultrapure water for 12 h, with the water changed every 4 h to remove residual DMSO and free copper ions. After dialysis, copper-loaded nano-assemblies or blank nano-assemblies solutions were obtained.

[0036] The particle size distribution was determined using dynamic light scattering (DLS). The average hydrated particle size of the copper-loaded nano-assemblies was approximately 10⁸ nm, and the polydispersity index (PDI) was 0.16. Figure 3 Transmission electron microscopy (TEM) observation showed that the nanoparticles were spherical, uniform in morphology, and showed no obvious aggregation. Figure 4 The results showed that N′-linolenico-N-methylthiobenzoylhydrazide could effectively coordinate copper ions and co-assemble with DSPE-PEG2000 to form stable copper-loaded nano-assemblies.

[0037] Example 3: In vitro stability test of copper-loaded nano-assemblies To verify the stability of the copper-loaded nanoparticle self-assemblies, the nanoparticles were stored at 25°C, and the particle size was measured using a dynamic light scattering instrument at different time points. Four parallel samples were measured each time, and the results are as follows: Figure 5 As shown.

[0038] The results showed that the copper-loaded nano-assemblies remained stable within 72 h, with a hydration kinetic diameter not exceeding 200 nm, demonstrating their good formulation stability.

[0039] Example 4: In vitro cytotoxicity experiments of different drugs The cytotoxic effects of different treatments on MC38 tumor cells were investigated using the CCK8 assay, with free copper chloride as a reference. Results are as follows: Figure 6 As shown in Table 1.

[0040] Table 1. Determination of cell viability 72 hours after drug treatment (IC50) 50 (μM)

[0041] The results in Table 1 show that the copper-loaded nano-assemblies prepared in Example 2 have good antitumor activity, which is significantly higher than that of free copper chloride.

[0042] Example 5: Antitumor effect of copper-loaded nano-assemblies in a mouse MC38 subcutaneous tumor model Construction of MC38 subcutaneous tumor model: MC38 cells were digested with trypsin, washed with PBS, and a cell suspension was prepared. After counting, the cells were divided into groups of 1×10⁶ cells per cell. 6 Cells were subcutaneously injected into the right abdominal wall of healthy male C57BL / 6 mice at a density of 100 μL per mouse. Tumors were allowed to grow to approximately 100 mm in size. 3 At that time, the mice were randomly divided into 4 groups (n=8).

[0043] To evaluate the antitumor activity of copper-loaded nanoassemblies, copper-loaded nanoassemblies (2 mg / kg based on copper content) were intravenously injected on days 0, 2, and 4, with copper chloride, blank nanoassemblies, and physiological saline serving as controls. Tumor volume was measured and recorded every two days. The longest diameter (L, mm) and shortest diameter (W, mm) of the tumor were measured using calipers, and V was calculated using the formula V = (L × W) / (L × W). 2 The tumor volume was calculated as W / 2 (W < L). The weight changes of each group of mice were recorded using an electronic scale.

[0044] Anti-tumor efficacy results such as Figure 7 As shown, compared with any control group, copper-loaded nano-assemblies significantly inhibited the growth rate of tumor volume in mice. Meanwhile, as... Figure 8 As shown, the copper-loaded nano-assemblies did not cause a significant decrease in mouse body weight, indicating that they have good in vivo safety.

Claims

1. A copper-loaded nano-self-assembled structure, characterized in that, The copper-loaded nano-assemblies are formed by the self-assembly of the compound N′-linaloyl-N-methylthiobenzoylhydrazine or its pharmaceutically acceptable salt, as shown in formula (I), with copper ions after coordination. ， (Ⅰ)。 2. The copper-loaded nano-self-assembled structure according to claim 1, characterized in that, The copper ions are derived from pharmaceutically acceptable copper salts or solvates.

3. The copper-loaded nano-self-assembled structure according to claim 2, characterized in that, The copper salt is selected from one or more of copper chloride and copper acetate.

4. The copper-loaded nano-self-assembled structure according to claim 1, characterized in that, N′-Linyl-N-methylthiobenzoylhydrazide was prepared by acylation reaction of linolenic acid chloride with N-methylthiobenzoylhydrazide.

5. A method for preparing the copper-loaded nano-self-assembled structure according to claim 1, characterized in that, Copper salts were dissolved in an aqueous phase to obtain a copper-containing aqueous phase; the compounds N′-linolenico-N-methylthiobenzoylhydrazide and DSPE-PEG shown in formula (I) were dissolved in an organic solvent to obtain a first organic solution and a second organic solution, and then the first organic solution and the second organic solution were mixed and injected into the copper-containing aqueous phase to form a copper-loaded nano self-assembled body. ， (Ⅰ)。 6. The preparation method according to claim 5, characterized in that, The copper salt is a pharmaceutically acceptable copper salt or its solvation; the copper salt is selected from one or more of copper chloride and copper acetate; The organic solvent contains dimethyl sulfoxide.

7. The preparation method according to claim 5, characterized in that, The molar ratio of copper ions, N′-linolenic acid-N-methylthiobenzoylhydrazine, and DSPE-PEG is 1~50:1~50:0.1~1.

8. Use of the copper-loaded nano-self-assembled structure according to any one of claims 1-4 in the preparation of a medicament for treating tumors, wherein the tumor is colon adenocarcinoma.

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